Synthetic genes and bacterial plasmids devoid of cpg

ABSTRACT

The invention relates to a new series of bacterial plasmid vectors which are fully devoid of CpG and which can express synthetic genes which do not contain CpG in the bacteria  Escherichia coli.

FIELD OF THE INVENTION

[0001] The present application relates to synthetic genes and toplasmids entirely devoid of CpG.

TECHNOLOGICAL BACKGROUND

[0002] Plasmids are genetic elements essentially found in bacteria, madeup of a molecule of deoxyribonucleic acid, which is most commonlycircular and the replication of which is autonomous and independent fromthat of genomic DNA. Natural plasmids isolated from a very broad varietyof bacteria are capable of accomplishing several cellular functions. Thefirst, which is vital for all plasmids, is that responsible for theirreplication, generally carried out in a manner which is synchronizedwith replication of the genomic DNA and cell division. Besides theregion required for replication of the plasmid, all natural plasmidscarry genes which encode proteins, the function of which most commonlyremains unknown due to a lack of scientific investigation with regard tothese genes. The number of genes present on a plasmid determines thesize of this plasmid, the smallest natural plasmids containing only twoto three genes. The properties of plasmids attracted research scientiststo them very early on, to make them vehicles for transporting andexpressing genes in prokaryotic cells, as well as in eukaryotic cells.The very rapid progress observed in the fields of the molecular biologyof nucleic acids and proteins, over the past two decades, can in part beattributed to the exploitation of recombined plasmids constructed fromfragments of natural DNA of plasmid origin or other cellular DNAs, andeven chemically synthesized.

[0003] The four bases adenine (A), guanine (G), cytosine (C) and thymine(T) which constitute deoxyribonucleic acid (DNA) are distributed in 16dinucleotide configurations, namely CG, GC, TA, AT, CC, GG, TT, AA, TG,CA, AG, CT, AC, GT, GA and TC. Analysis of the qualitative distributionof the dinucleotides of the DNA of thousands of plasmids for whichsequences are known reveals that the 16 dinucleotides are always presentin all natural plasmids or plasmids constructed in the laboratory.However, analysis of the quantitative distribution of the dinucleotidesof plasmids shows great disparities which depend, only partly, on thepercentage of each one of the four bases of the DNA. Specifically,comparison of the frequencies observed for each one of the dinucleotideswith those of the frequencies calculated on the basis of a randomassociation between two bases, for a given plasmid, can demonstratemajor differences for several dinucelotides in terms of an overrepresentation, or, on the contrary, an under representation (CampbellA., Mrazek J. and Karlin S. (1999) Proc Natl Acad Sci USA 96, 9184-9).The differences observed in the distribution of certain dinucleotides,not always the same, of natural plasmids isolated from bacteria ofphylogenically distant species have been explained by differences inspecificity in the mechanisms of repair, recombination and replicationacting on the cellular DNAs.

[0004] Gene transfers in vitro into cells in culture and in vivo intovarious animals are common practices undergoing great development, onthe one hand, for the purpose of gaining a better understanding of cellfunction and, on the other hand, in order to apply these techniques tocell and gene therapies. None of the viral vectors and plasmid vectorsamong the panoply of vectors available for gene transfer in animals hastaken a decisive advantage over the others, since each one hasadvantages, but also disadvantages. There is, however, an application inwhich naked plasmid DNA or plasmid DNA complexed with various substancesto facilitate DNA transport to the nucleus is the subject of intenseresearch activity, namely that of immunizing DNA. The principle ofimmunizing DNA is based on the immune responses observed in laboratoryanimals treated, by intramuscular or intradermal injection or byinhalation, with plasmid DNA encoding an antigenic peptide. It is nowwell established that a first consequence of introducing a plasmid DNAderived from the bacteria E. coli into the body of an experimentalanimal intravenously and intramuscularly is the rapid production ofvarious cytokines by the guard cells of the immune system (Krieg A. M.and Kline J. N. (2000) Immunopharmacology 48, 303-305). This response isextremely specific for bacterial DNA since DNA extracted from animalcells does not cause such an induction of cytokines under the sameconditions. The cellular mechanisms involved in this immune response arefar from being fully understood. However, it is known that therecognition which discriminates between bacterial DNA and DNA of animalorigin takes place at the level of structural differences relating tothe methylation of certain cytosines of the molecule. Specifically,mammalian DNA is naturally methylated at the cytosine of all CGdinucleotides (subsequently written CpG), with the exception of shortregions of high CpG density, called CpG islands, present in functionalregions in some promoters. DNA extracted from E. coli does not exhibitthis type of methylation due to the absence of the enzyme activitycapable of accomplishing this modification in this bacterium. It is,however, possible to methylate the CpGs of plasmid DNA extracted from E.coli in a test tube with an appropriate enzyme. Under these conditions,DNA methylated in vitro loses a great deal of its immunostimulantactivity compared to control nonmethylated DNA. The E. coli strain K12,from which virtually all the mutant strains used to produce plasmid DNAsare derived, contains an enzyme activity (DNA methylase dcm (Palmer B.R. and Marinus M. G. (1994) Gene 143, 1-12)) which leads to methylationof cytosine occurring in the nucleic acid context CC(A/T)GG. All plasmidvectors for gene transfer contain this sequence in varying number and,as a result, their DNA molecule contains methylated cytosines which arenot found in mammalian DNA. This form of methylation specific to E. colithus introduces another difference into the cytosine methylationsbetween bacterial DNA and that of mammals, which might contribute to theimmunostimulant capacity of plasmid DNA.

[0005] The CpG frequency in primate and rodent DNAs is, overall, muchlower than that expected on the basis of the frequency of cytosines andguanines. The CpG deficiency is dependent, for a given DNA fragment, onthe biological role of this fragment, intergenic regions containing onlya fifth of the expected frequency, while exons have a less markeddeficiency and, at the other extreme, some promoters containing a largeCpG island exhibit a CpG percentage close to that expected. Analysis ofthe data from sequencing human cDNAs and chromosomes reveals, however,broad heterogeneities in the CpG frequency for promoter regions andcDNAs. This observation is illustrated by the cDNA of the human geneencoding interleukin 2, which has just one CpG. Similarly, a portion ofthe promoter of this gene containing the TATA box does not contain anyCpG, but, on the other hand, the upstream portion rich in transcriptionfactor recognition sites contains CpGs. The regions positioned 3′ of thegenes, formed by the 3′ UTRs (untranslated regions) and thepolyadenylation and end of transcription sequences are rather poor inCpG. In humans, it is not unusual to find regions immediately downstreamof the genes, which are devoid of CpG. However, the human sequencingdata available at the end of 2000 have not made it possible todemonstrate a single transcriptional unit, made up of the transcriptionpromoter regions, a gene with or without an intron and thepolyadenylation region, which is completely devoid of CpG. The situationof the CpGs in E. coli is quite different from that of animal cellssince the frequency of CpGs in the genomic DNA of this bacterium isslightly greater compared to the calculated frequency. The same is truefor the CpGs of natural plasmids isolated from hospital strains of E.coli. The recombined plasmids resulting from genetic manipulations, usedfor gene transfer, exhibit variations in their CpG numbers which dependon the origin of the fragments inserted into the vector. Analysis of thesequences of several tens of recombined E. coli plasmids randomly takenfrom the GenBank databank shows that the plasmids most lacking in CpGhave, at the very most, a 50% deficiency in the number of their CpGs.

[0006] As regards the present invention, it provides products andmethods for synthesizing plasmid DNA in E. coli which is completelydevoid of CpG and in which the cytosines placed in the context CC(A/T)GGare not methylated. To the applicant's knowledge, this is the firstdescription of such products which exhibit such a structure while at thesame time having conserved their function.

DESCRIPTION OF THE INVENTION

[0007] The present invention provides means for producing plasmids whichare functional in a prokaryotic organism such as Escherichia coli, butwhich are nevertheless completely devoid of CpG. More particularly itprovides means for producing plasmids which are completely devoid ofCpG, and which are also free of cytosine methylation in the nucleic acidcontext CC(A/T)GG.

[0008] The present application thus relates to methods for producingsuch plasmids, and also to the elements constituting these plasmids,namely genes devoid of CpG which can be expressed in E. coli, promotersdevoid of CpG which are suitable for the expression of said genes, andorigins of replication devoid of CpG which are suitable for thebacterial transformation of said plasmids. The present application isalso directed towards the biotechnological and medical applications ofthese products. Each one of these products has the particularcharacteristic of being completely devoid of CpG, while at the same timehaving conserved its functionality in a prokaryote such as E. coli. Thepresent application also provides an E. coli strain specially suited tothe production of the plasmids according to the invention, this strainhaving the particular characteristic of allowing stable replication ofthese plasmids and of the genetic material which they transport, withoutimpairing their function, and without, however, inducing methylation atCC(A/T)GG sites (strain comprising an inactivated dcm gene).

[0009] One of the common concepts linking the various aspects of theinvention is therefore to make it possible to produce plasmids which arecompletely devoid of CpG and which have, despite everything, conservedtheir functional properties in a prokaryote such as E. coli. To theapplicant's knowledge, this is the first description of such means.

[0010] The present application is thus directed toward a method forproducing a plasmid which is a vector of at least one gene, and which iscompletely devoid of CpG characterized in that a plasmid is constructedby assembling, by enzyme ligation, DNA fragments, all devoid of CpG,corresponding to an origin of replication for the plasmid and toelements constituting a transcriptional unit for said at least one gene,and in that this plasmid is transferred into an Escherichia coli strainexpressing the pi protein for replication of the plasmid.

[0011] Plasmids isolated from wild-type bacterial strains generallyaccomplish three functions in relation to replication, namely initiationof DNA replication, control of replication and stable maintenance of theplasmid during successive divisions. Plasmids constructed in thelaboratory do not always exhibit all of these functions. The number ofcopies of the plasmids is, for example, quite often increased comparedto the parent plasmid, denoting that replication control elements havebeen modified. The plasmid R6K contains three origins of replication,alpha, gamma and beta, linked on the same DNA fragment (Filutowicz M.and Rakowski S. A. (1998) Gene 223, 195-204). Each one of the origins isactivated by the R6K specific pi initiation protein encoded by the R6Kpir gene. In order to be functional, the three origins need a 277 bpsequence, known by those skilled in the art as “core”, located at thecenter of the fragment carrying the three origins, and also anadditional single fragment positioned in cis, i.e. present on the sameDNA molecule. When the sequences of the alpha and beta origins aredeleted, the remaining gamma origin allows autonomous duplication of theplasmid on the condition that the pir gene is present in cis on theplasmid or in trans on the chromosome of the bacterium. The inventorschose to focus more particularly on the smallest of the three origins,namely the gamma origin, which has the advantage of containing all theelements required for controlled replication of the plasmid, namely thecore and an adjacent activating sequence. The core is made up of a piprotein-binding sequence repeated 7 times, and an AT-rich sequence. Theactivating region contains binding sites for several cellular proteinsof the bacterium, required for stably maintaining the plasmid. Thenumber of copies of the plasmids containing only the gamma origindepends on the pi protein; mutant forms of pi leading to a largeincrease in the number of copies of the plasmid have been isolated andcharacterized. As shown in greater detail in the examples below, theinventors have succeeded in constructing, from the gamma origin ofreplication of the plasmid R6K, origins of replication which no longerexhibit any CpG, while at the same time having conserved theirfunctionality intact. It may be noted that the specific choice of R6Kgamma as starting material, namely the choice of a small replicon whichexhibits only a small number of CpG, is a particularly relevant choiceinsofar as, when other plasmids such as those of the pUC series are usedas starting material, CpG-free plasmids which remain functional are notsuccessfully obtained: all the attempts made by the inventors tochemically reconstitute the minimum pUC sequence by replacing thecytosines of CpGs with guanine or adenine resulted in DNA fragmentswhich had lost all functional replication activity. As regards theplasmids which comprise a CpG-free origin of replication according tothe invention, they have conserved their ability to replicate stablywithin a prokaryote cell such as E. coli, and E. coli K12 in particular,provided, of course, that they are provided with the pi protein requiredto activate the replication (wild-type pir or mutated pir such as pir116 in cis or in trans). An origin of replication for plasmid accordingto the invention is characterized in that its sequence corresponds tothat of the R6K gamma origin of replication in which each G of the CpGsof the repeat-region of the core has been replaced with an A, a C or aT, or each C of the CpGs has been replaced with a G, an A or a T.Various CpG-free origins of replication have thus been obtained, which,surprisingly, are still capable of performing the functions of origin ofreplication of plasmids in E. coli and, what is more, are capable ofperforming these functions for genes and transcriptional units which arethemselves devoid of CpG. The examples below give some illustrationsthereof (cf. origins R6K gamma M2A, R6K gamma M2C, R6K gamma M2T inexamples 7-10). The present application is directed more particularlytoward any origin of replication whose sequence comprises the sequenceSEQ ID No. 12 or SEQ ID No. 13 (FIGS. 12 and 14). It has also beendemonstrated that the pi protein-binding sequence can be not repeated 7times, as is observed in the standard R6K gamma origin with CpG, butthat the number thereof can be limited to 5 or 6, without howeverimpairing the functions of the origin of replication. The presentapplication is thus directed toward any origin of replication accordingto the invention as defined above, which would comprise only 5 or 6repeats of the pi protein-binding sequence. By virtue of these CpG-freefunctional origins of replication, the inventors have been able toconstruct various plasmids which, notably, have conserved theirtransfection vector functions.

[0012] The creation of E. coli plasmids devoid of CpG necessarilyrequires having functional genes (which can be expressed in prokaryotessuch as E. coli) which do not contain any CpG. Thus, selection of thebacteria transformed with a recombined plasmid DNA involves a gene whoseprotein confers a dominant advantage on the bacterium. Most commonly,the selective marker is introduced by a gene for resistance to anantibiotic which is active on the E. coli bacterium. Analysis of thewide variety of resistance genes used in E. coli shows that, withoutexception, they all contain CpGs, very often in very high numbers forresistance genes originating from Streptomyces which produce theantibiotic for selection. Similarly, it is necessary to have reportergenes which are CpG-free while at the same time remaining functional.

[0013] Analysis of several hundred chromosomal and plasmid genes of theE. coli bacterium, the well-characterized sequences of which areavailable in several databanks, reveals that all genes greater than 250pb in size, without exception, consist of 16 dinucleotides.

[0014] The present invention demonstrates that it is, despiteeverything, possible to construct genes which are functional in E. coliand which are devoid of CpG. The inventors have in fact developed amethod for obtaining genes devoid of CpG while at the same time beingable to be expressed in E. coli. This method is based on the synthesisof a polynucleotide chain by following the amino acid chain of a proteinwhich can be expressed in E. coli, assigning to each amino acid anucleotide codon chosen from those which, according to the genetic code,and taking into account the degeneracy of this code, correspond to thisamino acid, but eliminating from this choice:

[0015] i. all codons containing a CpG in their sequence: this concernsthe codons ACG (Thr), CCG (Pro), GCG (Ala), TCG (Ser), CGA (Arg), CGC(Arg), CGG (Arg) and CGT (Arg), and

[0016] ii. codons which finish with a C when the codon which follows itdirectly begins with a G. Examples of a gene thus obtained comprise theNeoΔCpG gene (SEQ ID No 316; cf. example 11).

[0017] According to one variant of implementation of the invention, thecodons for which the frequencies are low in proteins of human originwill also be eliminated from said choice: this concerns the codons ATA(Ile), CTA (Leu), GTA (Val) and TTA (Leu). The set of possible codonstherefore, according to this variant, corresponds to the following set:

[0018] GCA (Ala), GCC (Ala), GCT (Ala), AGA (Arg), AGG (Arg), AAC (Asn),AAT (Asn), GAC (Asp), GAT (Asp), TGC (Cys), TGT (Cys), CAA (Gln), CAG(Gln), GAA (Glu), GAG (Glu), GGA (Gly), GGC (Gly), GGG (Gly), GGT (Gly),CAC (His), CAT (His), ATC (Ile), ATT (Ile), CTC (Leu), CTG (Leu), CTT(Leu), TTG (Leu), AAA (Lys), AAG (Lys), TTC (Phe), TTT (Phe), CCA (Pro),CCC (Pro), CCT (Pro), TCA (Ser), TCC (Ser), TCT (Ser), AGC (Ser), AGT(Ser), ACA (Thr), ACC (Thr), ACT (Thr), TAC (Tyr), TAT (Tyr), GTC (Val),GTG (Val), GTT (Val),

[0019] to which rule ii. above should of course be applied. Examples ofa gene obtained in accordance with this variant of implementationcomprise in particular the LacZΔCpG gene (positions 3 to 3056 of SEQ IDNo 9; cf. example 5).

[0020] Preferably, said choice of codon will also be made so as to avoidstructures which are unfavorable for the messenger RNA, such as thepresence of splice sequences, of direct or inverted repeat sequences, ofstem-loop structures or of polyadenylation signals. The number and thesize variety of the genes synthesized by this method are illustrated inthe examples below, which show that it is thus possible to envision thesynthesis of genes completely devoid of CpG which nevertheless remainfunctional in E. coli. As a reference protein, any protein which can beexpressed by E. coli can be chosen, for example a protein encoded by agene for resistance to an antibiotic, such as the genes for resistanceto zeocin® (phleomycin), to hygromycin, to blasticidin or to puromycin,or a protein encoded by reporter genes such as lacZ.

[0021] The present application is also directed toward such a method forobtaining genes devoid of CpG, which can be expressed in E. coli, andalso any gene of at least 250 bp which can be obtained using thismethod. More particularly, the present application is directed toward:

[0022] any gene the sequence of which comprises the sequence SEQ ID No.1 from position 3 to position 374 (FIG. 1), the sequence SEQ ID No. 3from position 3 to position 1025 (FIG. 3), the sequence SEQ ID No. 5from position 3 to position 422 (FIG. 5), the sequence SEQ ID No. 7 fromposition 3 to position 599 (FIG. 7), and also any use of these genes asselection markers, and

[0023] any gene the sequence of which comprises the sequence SEQ ID No.9 from position 3 to position 3056 (cf. FIG. 9), and any gene thesequence of which comprises the sequence SEQ ID No. 316 (positions 3 to797 of the DNA sequence presented in FIG. 18 (encoding SEQ ID No. 317)),and also any use of such a gene as a reporter gene.

[0024] The expression of a plasmid gene also requires having promoterssuitable for the cell harboring the plasmid. The fact that the E. coligenome has been entirely known for a few years has facilitated the studyof various noncoding elements exhibiting specific functions. Resultsfrom studies relating to the nature of the E. coli promoters arecontinually being updated and made public on the PromEC site accessiblevia the Internet (http://bioinfo.md.huji.ac.il/marg/promec). An analysisof the 471 well-defined promoters from base −75 to +25 relative to thetranscription initiation point +1 reveals that only 6 of them do notpossess CpG. The addition of each one of the 6 promoters synthesizedchemically and placed upstream of the lacZ gene encoding E. coliβ-galactosidase has proved to be negative for detection of the activityof this reporter gene. The lack of strong homology with the consensussequences of the −10 and −35 canonic boxes suggests that these promotersare low strength promoters and, alternatively, that said promoters mightbe regulated by induction conditions yet to be defined for each one ofthem. An analysis of the well-characterized promoters of E. coli hasrevealed that only about 10 do not contain any CpG in the specific boxesfor recognition by RNA polymerase. A bibliographical search has alsorevealed that these promoters are all inducible by various stimuli, asituation which is sometimes desired but most often relinquished forexpression which is constitutive in nature. As regards the inventors,they have succeeded, by random PCR assembly of fragments exhibitingshort consensus sequences devoid of CpG, drawn from several strongpromoters, in developing novel promoters which are suitable for theexpression of genes without CpG in E. coli, and which have theparticular advantage of being very strong constitutive promoters and ofbeing completely devoid of CpG. Example 6 below illustrates theconstruction of the novel promoter EM2K using this technology. Thepresent application is more particularly directed toward any promoter,the sequence of which comprises the sequence SEQ ID No. 11 (cf. FIG.11).

[0025] The characterized transcription terminators in E. coli are madeof short sequences, several of which do not possess any CpG, and theinventors have been able to verify that such terminators effectivelyperform their function when they are associated, in E. coli, with aCpG-free promoter and gene according to the invention.

[0026] The present application is thus directed toward anytranscriptional unit which comprises at least one CpG-free geneaccording to the invention, and at least one CpG-free promoter accordingto the invention. Such a transcriptional unit may also comprise at leastone CpG-free terminator. The invention thus provides, for the firsttime, a nucleotide group which is completely devoid of CpG, and whichcan nevertheless be expressed in E. coli, performing its normalfunctions therein.

[0027] The present application is thus directed toward any plasmid whichcomprises an origin of replication according to the invention. Suchplasmids may also comprise a CpG-free gene according to the inventionand/or a CpG-free promoter according to the invention and/or a CpG-freetranscription terminator, or a transcriptional unit according to theinvention. The plasmids according to the invention therefore have theadvantage of being able to exhibit no CpG in their structure, while atthe same time still being capable of performing expression vectorfunctions. Examples of such plasmids are given in the examples below.

[0028] The present application is more particularly directed toward anyplasmid of SEQ ID No. 14 (FIG. 15).

[0029] Any cell transformed with at least one element selected from thegroup consisting of the CpG-free genes according to the invention, theCpG-free promoters according to the invention, the CpG-free origins ofreplication according to the invention and the CpG-free plasmidsaccording to the invention also falls within the field of the presentapplication. Such a cell according to the invention may also comprise agene encoding a pi protein, such as wild-type pir or mutated pir, pir116. Advantageously, a transformed cell according to the invention is anE. coli cell.

[0030] To replicate a sufficient number of functional copies of aplasmid according to the invention, those skilled in the art have attheir disposal many bacteria, such as, for example, E. coli K12 bacteriawhich are conventionally used for the purposes of plasmid replication.The original K12 strain of E. coli has a DNA methylase which introducesa methyl group onto all the cytosines placed in the context CC(A/T)GG ofthe genomic and plasmid DNAs of the bacterium. All of the variousstrains of the K12 line have this activity due to a methylase encoded bythe dcm gene (Palmer B. R. and Marinus M. G. (1994) Gene 143, 1-12).Since methylation of the plasmid DNAs prepared from dcm⁺ strains of E.coli leads to a modification of the DNA molecule which is undesirablefor the transfer of genes into eukaryote cells, the inventors havedeveloped a strain which makes it possible both for plasmids with theCpG-free R6K gamma origin in accordance with the invention to function,and plasmid DNA devoid of methylation on the dcm sites to be obtained.For this, a new gene was constructed by the inventors, by deleting thedcm gene from position +3 after the ATG to position −14 before the TGAin the dcm gene of a pir 116 strain (cf. Example 10 below).

[0031] The dcm gene is located in a chromosomal region the sequence ofwhich can be obtained via GenBank with the accession number D90835(cloneKohara #344: 43.5-43.9 min).

[0032] A deletion in the gene (−) introduces the additional advantage ofavoiding any reversion of the gene to the wild-type form. dcm⁻¹ mutantstrains have thus been produced by the inventors; they exhibit nonegative phenotype which might impair bacterial growth or modify thequality and quantity of the plasmid DNA. More particularly, an optimizedstrain of E. coli has been constructed by targeted inactivation of thedcm gene using a parent strain expressing a mutated pi protein at a siteleading to an increase in the number of copies of CpG-free plasmids.This optimized strain allows quality and abundant production of theplasmid DNAs which are the subjects of this invention, and which aredevoid of CpG and free of methylation on the cytosines of the dcm sites.The present application is therefore directed toward any cell comprisinga gene encoding the pi protein, which is transformed with the deleteddcm gene according to the invention, and any method of replicatingplasmids, which comprises transforming such a cell with a plasmid, andculturing the transformed cell under conditions suitable for replicationof this plasmid.

[0033] The present application is thus directed toward a method forproducing a plasmid completely devoid of CpG and free of methylation oncytosine in the nucleic acid context CC(A/T)GG, characterized in that aplasmid according to the invention is produced by replication in anEscherichia coli strain expressing the pi protein, which is deficientfor the dcm methylation system.

[0034] Any kit for producing plasmids, which comprises at least one cellaccording to the invention, also falls within the field of the presentapplication. These kits are particularly suitable for the replication ofplasmids according to the invention, in order to avoid these plasmids,the structure of which is devoid of CpG, being, moreover, methylated onCC(A/T)GG during their replication.

[0035] The invention thus provides a complete set of transformationmeans devoid of CpG; CpG-free genes, CpG-free promoters, CpG-freetranscriptional units, CpG-free origins of replication for plasmid,CpG-free plasmids, cells specially suited to replication of plasmidswithout methylation of cytosines. These novel means find directapplications for genetic transformation of cells for biotechnological ormedical purposes. Such products are in fact exceptionally well suited tothe production of DNA vaccine compositions intended for humans oranimals.

[0036] The invention is illustrated by the following examples, in whichreference is made to the figures:

[0037]FIG. 1: Sequence of the Sh ble ΔCpG gene (CpG-free),

[0038]FIG. 2: List of the oligonucleotides used for assembling the Shble ΔCpG gene,

[0039]FIG. 3: Sequence of the Hph ΔCpG gene,

[0040]FIG. 4: List of the oligonucleotides used to assemble the Hph ΔCpGgene,

[0041]FIG. 5: Sequence of the Bsr ΔCpG gene,

[0042]FIG. 6: List of the oligonucleotides used to assemble the Bsr ΔCpGgene,

[0043]FIG. 7: Sequence of the Pac ΔCpG gene,

[0044]FIG. 8: List of the oligonucleotides used to assemble the Pac ΔCpGgene,

[0045]FIG. 9: Sequence of the LacZ ΔCpG gene,

[0046]FIG. 10A: List of the oligonucleotides used to assemble the firstthird of the LacZ ΔCpG gene,

[0047]FIG. 10B: List of the oligonucleotides used to assemble the secondthird of the LacZ ΔCpG gene,

[0048]FIG. 10C: List of the oligonucleotides used to assemble the thirdthird of the LacZ ΔCpG gene,

[0049]FIG. 11: Sequence of the EM7 promoter (1−), and of thedegenerative oligonucleotides (2−) used to construct the EM2K promoterdevoid of CpG (3−),

[0050]FIG. 12: Sequence of the R6K gamma M2A origin of replication,

[0051]FIG. 13: List of the oligonucleotides used to assemble the R6Kgamma M2A origin of replication,

[0052]FIG. 14: Sequence of the R6K gamma origin of the plasmidpGTR6Kneoc9 delimited by the PacI sites,

[0053]FIG. 15: Sequence of the plasmid pSh-LacZΔCpG gene,

[0054]FIG. 16: Map of the plasmid pShΔCpG,

[0055]FIG. 17: Map of the plasmid pSh-LacZΔCpG,

[0056]FIG. 18: Sequence of the NeoΔCpG gene (CpG-free) [position 3 to797 of DNA sequence ═SEQ ID No. 316; protein sequence ═SEQ ID No. 317),

[0057]FIG. 19: Sequence of the oligonucleotides SEQ ID No. 318 to SEQ IDNo. X used to assemble the NeoΔCpG gene.

EXAMPLE 1 Construction of the Sh ble Gene for Resistance to ZeocinDevoid of CpG

[0058] The Sh ble ΔCpG gene, the sequence of which is given in FIG. 1(positions 3 to 377 of SEQ ID No. 1) was synthesized from an assembly ofoverlapping oligonucleotides (20-40 pb in size), the sequences of whichare given in FIG. 2. The assembly method is carried out in three steps;the first step consists of phosphorylation of the oligonucleotides ofthe coding strand, in a second step, all the oligonucleotides of bothstrands are combined by hybridization and ligation and, in the finalstep, the gene is amplified by PCR. This method was successfully used tosynthesize all the synthetic genes mentioned in Examples 1, 2, 3, 4 and5. Details of the method are given for the Sh ble ΔCpG gene:

[0059] The 10 oligonucleotides, from OL26199 to OL27099 (FIG. 2),corresponding to the coding strand, are phosphorylated according to thefollowing procedure: 1 μl of each one of the oligonucleotides taken upin water at 250 μM are mixed in a microtube containing 15 μl of water soas to bring the final solution to a concentration of 100 picomol permicroliter. 5 μl of this solution are then mixed with 10 μl of0.10-times concentrated polynucleotide kinase buffer, 0.4 μl of a 50 mMATP solution, 85 μl of water and 1 μl of the enzyme (at 10 μ/μl), andthe entire mixture is incubated for 4 hours at 37° C. (solution A).

[0060] A solution of the oligonucleotides of the noncoding strand ismade up by mixing 1 μl of each oligonucleotide (OL27199 to OL28199; cf.FIG. 2) and 1 μl of the oligonucleotide OL26099 (FIG. 2), to whichsolution 43 μl of water are added in order to obtain a final solution at54 picomol per μl (solution B).

[0061] Assembly of the gene is carried out first by mixing 10 μl ofsolution A, 1 μl of solution B, 6 μl of a 100 mM KCl solution, 3 μl of a0.5% NP-40 solution, 4 μl of a 50 mM MgCl₂ solution, 3 μl of a 10 mM ATPsolution and 7.5 μl of Pfu ligase (30 units), and the mixture is thenheated in a programmable thermocycler for 3 minutes at 95° C. and then 3minutes at 80° C., before undergoing 3 cycles of one minute at 95° C.,followed by a change from 95° C. to 70° C. in 1 minute, and then achange from 70° C. to 55° C. in 1 hour and, finally, 2 hours at 55° C.The mixture of the assembled oligonucleotides is then amplified with theprimers OL26099 and OL27199. The amplification product is purified on apromega column, digested with the NcoI and NheI restriction enzymes, andcloned into the plasmid pMOD1LacZ(wt) linearized with NcoI and NheI. Thesequences of the plasmid DNA of 2 zeocin-resistant clones which appearedafter transformation of the E. coli strain GT100 (available fromInvivogen) with the mixture of the ligation between the vector fragmentand the PCR fragment, were found to be in accordance with the desiredsequence given in FIG. 1. This synthetic gene, placed under the controlof the bacterial EM7 promoter (vector pMOD1Sh ΔCpG) confers a zeocinresistance identical to that provided by the same vector containing thenative Sh ble gene with the recipient E. coli strain GT100.

EXAMPLE 2 Construction of the Hph Gene for Resistance to HygromycinDevoid of CpG

[0062] The synthetic Hph ΔCpG (sequence SEQ ID No. 3 given in FIG. 3)was constructed according to the method described in Example 1. The twostrands were synthesized using oligonucleotides of 60 bases plus twooligonucleotides of 30 bases with an overlapping region of 30 bases. Theassembly of the various oligonucleotides given in FIG. 4 was carried outthrough a final PCR with the sense oligonucleotide TTCAGCTGAGGAGGCACATC(SEQ ID No. 299) and the reverse oligonucleotide CTCAGGATCCGCTAGCTAAT(SEQ ID No. 300) according to the experimental conditions mentioned inthe preceding example.

[0063] The amplified and purified fragment (1068 pb) was then digestedwith the BspHI and NheI restriction enzymes and cloned into the vectorpMOD2 LacZ(wt), in which the NcoI site of pMOD1 is replaced with theBspHI site. The E. coli clones containing this recombinant vector wereselected on FastMedia™ Hydro Agar medium (Cayla). The sequence SEQ IDNo. 3 in FIG. 3 was confirmed by sequencing on both strands of theplasmid DNA of two hygromycin-resistant clones. This synthetic gene,placed under the control of bacterial EM7 promoter (vector pMOD2 HphΔCpG), confers a hygromycin B resistance which is at least equal to thatprovided by the same vector containing the native Hph gene with therecipient E. coli strain GT100.

EXAMPLE 3 Construction of the Bsr Gene for Resistance to BlasticidinDevoid of CpG

[0064] The Bsr ΔCpG gene, the sequence of which is given in FIG. 5 (SEQID No. 5), was synthesized using the oligonucleotides indicated in 6, byfollowing the method described in Example 1. The mixture of theassembled oligonucleotides was amplified with the primers OL64 and OL76(cf. FIG. 6). The amplified and purified fragment was then digested withthe BspHI and NheI restriction enzymes and cloned into the vector pMOD2LacZ(wt). The E. coli clones containing this recombinant vector wereselected on FastMedia™ Blasti Agar medium (Cayla). The sequence SEQ IDNo. 5 in FIG. 5 was confirmed by sequencing on both strands of theplasmid DNA of two blasticidin-resistant clones.

[0065] This synthetic gene, placed under the control of the bacterialEM7 promoter (vector pMOD2 Bsr ΔCpG), confers resistance to blasticidinwhich is identical to that provided by the same vector containing thenative Bsr gene with the recipient E. coli strain GT100.

EXAMPLE 4 Construction of the Pac Gene for Resistance to PuromycinDevoid of CpG

[0066] The BspHI-NheI fragment (Pac ΔCpG gene; SEQ ID No. 7), thesequence of which is given in FIG. 7, was synthesized by assembling theoligonucleotides indicated in FIG. 8.

[0067] The mixture of the assembled oligonucleotides was amplified withthe sense primer pur24 (AGGACCATCATGACTGAG; SEQ ID No. 301) and thereverse primer pur25 (ATCATGTCGAGCTAGCTC; SEQ ID No. 302). The purifiedBspHI-NheI fragment was cloned into the plasmid pMOD2LacZ (wt) betweenthe BspHI and NheI sites. The sequences of the plasmid DNA of 2puromycin-resistant clones of the GT100 strain, which appeared on theFastMedia™ puro Agar medium (Cayla) after transformation by the productof the ligation between the vector fragment and the PCR fragment, werefound to be in accordance with the desired sequence given in FIG. 7. Thesynthetic Pac ΔCpG gene, placed under the control of the bacterial EM7promoter (vector pMOD2 Pac ΔCpG), confers a puromycin resistance whichis slightly greater than that provided by the same vector containing thenative pac gene with the recipient E. coli strain GT100.

EXAMPLE 5 Construction of the LacZ Gene Devoid of CpG Encodingβ-Galactosidase of E. coli

[0068] The synthetic LacZ ΔCpG gene (SEQ ID No. 9 given in FIG. 9) wasconstructed according to the method described in the preceding examples.Given the size of the gene to be produced (more than 3000 pb), theconstruction was carried out in 3 distinct parts, conserving the EcoRVand SacI restriction sites at the same sites as on the native sequenceof the lacZ gene. For each part, the two strands were synthesized usingoligonucleotides of 40 bases plus two oligonucleotides of 20 bases withan overlapping region of 20 bases.

[0069] The first region corresponds to the NcoI-EcoRV fragment (Part I),the second region corresponds to the EcoRV-SacI fragment (Part II) andthe third region corresponds to the SacI-NheI fragment. The assembly ofthe various oligonucleotides given in FIGS. 10A (oligonucleotides usedto assemble part I), 10B (oligonucleotides used to assemble part II),and 10C (oligonucleotides used to assemble part III) was carried out byPCR according to the same experimental conditions stated in thepreceding examples. The gradual cloning of the three parts of thesynthetic gene was carried out in the vector pMOD1 LacZ (wt). Thefunctionality of each cloned part and also that of the completesynthetic gene present on the vector pMOD1 LacZ was demonstrated byrevealing the β-galactosidase activity on FastMedia™ Amp Xgal Agarmedium (Cayla), of the recombinant clones obtained in the MC1061ΔLacstrain. The complete synthetic LacZ ΔCpG gene, placed under the controlof the EM7 promoter, gives 30% less β-galactosidase activity(luminometric assay of protein extracts from culture) compared to theexpression of the native LacZ gene in the same plasmid environment.

EXAMPLE 6 Construction of a Strong Constitutive Promoter for E. coli,Devoid of CpG

[0070] The bacterial EM7 promoter present on vectors of the pMOD1 typeis a synthetic promoter which is constitutive and strong in E. coli. Itssequence, which contains 3 CpG (SEQ ID No. 297 in FIG. 11), was used asa reference to produce a bacterial promoter devoid of CpG. We produced“linker” oligonucleotides which were degenerative at 4 places (indicatedW, D, W and H on the sequence SEQ ID No. 298 in FIG. 11) and compatiblewith the AseI and NcoI restriction sites. These various oligos werehybridized and cloned into pMOD1 ShΔCpG between the AseI and NcoIrestriction sites of the EM7 promoter. After selection of therecombinant clones on FastMedia™ Zeo Agar medium and determination ofthe promoter sequence of the most zeocin-resistant clone, we selectedthe EM2K promoter (sequence SEQ ID No. 11 in FIG. 11) as the bacterialpromoter devoid of CpG.

EXAMPLE 7 Synthesis of the R6K Gamma Origins Devoid of CpG

[0071] The PacI DNA fragment containing the R6K gamma M2A origin (SED IDNo. 12 in FIG. 12) was synthesized by PCR from the assembly of theoligonucleotides indicated in FIG. 13. The R6K gamma M2A fragmentassembly was amplified with the primers RK15(GCAGGACTGAGGCTTAATTAAACCTTAAAAC; SEQ ID No. 303) and RK16(AAGTCTCCAGGTTAATTAAGATCAGCAGTTC: SEQ ID No. 304), and the fragments,after digestion with the PacI enzyme, were cloned into a plasmid(pGTCMVneo) containing the kanamycin resistance gene and the pUC originof replication bordered by 2 PacI sites. Many transformants of the GT97strain (which expresses the pi protein) were analyzed and only clonescontaining a high-copy plasmid conserved after several rounds ofsubculturing in the absence of kanamycin were selected. Aftersequencing, it was found that the ori fragment of most of these plasmidscould have a lower number (5-6) of repeat sequences, instead of the 7,of the natural origin of the R6K plasmid. One of these novel sequencesof the synthetic R6K gamma origin devoid of CpG is given in SEQ ID No.13 in FIG. 14).

[0072] Two other versions of the R6K gamma origin, in which the G ofeach CpG present in the repeat sequences (22 bp element repeated severaltimes in the pi protein-binding region) has been replaced with a C, togive the origin (R6K gamma M2C), or a T, to give the origin (R6K gammaM2T), were synthesized in a similar manner. The functionality of thesenovel R6K gamma origins in which the G of the CpGs of the repeatsequences is replaced with a C or with a T, added to the example of theorigin of FIG. 13, in which the G is replaced with an A, demonstratesthat the CpGs of these repeat sequences do not play a role in thefunctionality of the origin.

EXAMPLE 8 Assembly of Plasmid Vectors Completely Devoid of CpG,Expressing a Gene for Resistance in E. coli

[0073] Firstly, a PacI-PacI cassette containing the bacterial EM2Kpromoter and the Sh ΔCpG zeocin resistance gene followed by a CpG-freebacterial terminator was prepared. For this, “linker” oligonucleotidescontaining the sequence of the t1 terminator of the intergenic regionrpsO-pnp of E. coli were hybridized and cloned between the NheI and PacIsites of the vector pMOD1 EM2K Sh ΔCpG: “linker” oligonucleotides:rpsO-1 (5′->3′): CTAGCTGAGTTTCAGAAAAGGGGGCCTGAGTGGCCCCTTTTTTCAACTTAATSEQ ID No. 305 rpsO-2 (5′->3′):TAAGTTGAAAAAAGGGGCCACTCAGGCCCCCTTTTCTGAAACTCAG. SEQ ID No. 306

[0074] The recombinant vector obtained (pMOD1 EM2K sH ΔCpG Term) wasverified by sequencing in the region of the terminator sequence whichdoes not naturally contain any CpG. The EM2K-Sh ΔCpG-Term cassettecontained in this vector was then amplified by PCR so as to flank thetwo sides with PacI sites using the following primers: PACI-UP (5′->3′):ATCGTTAATTAAAACAGTAGTTGACAATTAAACATTGGC SEQ ID No. 307 PACI-DOWN(5′->3′): ATCGTTAATTAAGTTGAAAAAAGGGGCC. SEQ ID No. 308

[0075] This amplified fragment was then purified and cleaved with PacI,and then assembled with PacI fragment containing the R6K gamma ΔCpGorigin described in Example 7. After transformation of this ligationmixture into the GT97 strain (which expresses the pi protein) andselection on FastMedia™ Zeo medium, analysis of the recombinant clonesobtained revealed two possible orientations of the PacI-PacI fragmentcontaining the R6K gamma ΔCpG origin. The orientation selected in pShΔCpG is represented in FIG. 16.

EXAMPLE 9 Assembly of a Plasmid Vector Completely Devoid of CpG,Expressing the Zeocin Resistance Gene and the β-Galactosidase Gene in E.coli.

[0076] The vector pSh ΔCpG described in Example 8 (FIG. 16) was used toinsert the synthetic LacZ gene devoid of CpG between the EcoRI and NheIsites. For this, EcoRI- and NcoI-compatible “linker” oligonucleotidescontaining a ribosome-binding site consensus sequence from E. coli werehybridized and cloned with the NcoI-NheI LacZΔCpG fragment of pMOD1LacZΔCpG, between the EcoRI and NheI sites of the vector pMOD1 EM2KShΔCpG. “linker” oligonucleotides used: rbs-1 (5′->3′):AATTCTGAGGAGAAGCT SEQ ID No. 309 rbs-2 (5′->3′): CATGAGCTTCTCCTCAG SEQID No. 310

[0077] Transformation of this ligation mixture into the GT97 strain(which expresses the pi protein) and selection on FastMedia™ Zeo Xgalmedium made it possible to obtain the recombinant clones containing thevector pSh-LacZΔCpG (FIGS. 15 and 17). This vector co-expresses, underthe control of the bacterial EM2K promoter, in an artificial operonsystem, the ShΔCpG and LacZΔCpG genes.

EXAMPLE 10 Production of an E. coli Strain Expressing the Mutant Proteinpi116 and Carrying a Deletion in the dcm Gene

[0078] The pir gene encoding the pi protein which is essential forinitiating replication of the R6K gamma origin, and also the mutatedgene pir116 which leads to an increase in the number of copies of R6Kgamma plasmids, have been introduced, in a functional form, into variousE. coli K12 strains by various groups. Strains of this type can beobtained from the E. coli Genetic Stock Center(http://cgsc.biology.yale.edu), and are also commercially available fromcompanies specializing in supplying biological material for research.This is the case, for example, of the pir1 (pir116) and pir2 (wild-typepir) strains provided by the company Invitrogen, whose products can bepurchased in all European countries. The GT97 strain of the K12 line,which has the genotype Δlac169 hsdR514 endAl recAl codBa uidA(ΔMlul)::pir 116 (available from InvivoGen), was chosen for itssimplicity, the consistency of the R6K gamma plasmid DNA preparationsand its high levels of competence, from several K12 pir strains ofdistinctive genotype for some genes. The introduction of a deletion intothe dcm gene of the GT97 strain was carried out in the following way:

[0079] Two DNA regions, of 1.8 kb and 1.5 kb, flanking respectively theATG initiation codon (fragment A) and the TGA stop codon (fragment B) ofthe dcm gene were amplified by PCR. The fragment A was then amplifiedwith the pair of primers OLdcmAF (TTTTGCGGCCGCTTGCTGCGCCAGCAACTAATAACG;SEQ ID No. 311) and OLdcmAR (CCTTGGATCCTGGTAAACACGCACTGTCCGCCAATCGATTC;SEQ ID No. 312) and fragment B was amplified with the pair of primersOLdcmBF (TTTTGGATCCTCAGCAAGAGGCACAACATG; SEQ ID No.313) and OLdcmBR(TTTTCTCGAGAAACGGCAGCTCTGATACTTGCTTC; SEQ ID No. 314). The restrictionsites for the NotI (GCGGCCGC), BamHI (GGATCC) and XhoI (CTCGAG) enzymeswere introduced into the primers in order to combine fragment A andfragment B with one another, forming a genetic element flanked by theNotI and XhoI sites. The region of the dcm gene is thus reconstituted,creating a deletion which stems from position +3 after the ATG toposition −14 before the TGA. This genetic element was cloned into pKO3(Link A. J., Phillips D. and Church G. M. (1997) J Bacteriol 179,6228-37), a vector developed for allele replacement in Escherichia coli,with thermosensitive replication, between the NotI and SalI sites, togive the plasmid named pKO3Δdcm. The GT97 strain was co-transformed withthis plasmid and with a plasmid which expresses the RecA protein(pFL352). A transformant containing the two plasmids was cultured at anonpermissible temperature (42° C.) in the presence of chloramphenicolin order to select clones which have integrated pKO3Δdcm into thebacterial chromosome by homologous recombination. A subclone resistantto chloramphenicol at 42° C. was then cultured at 30° C. on a mediumcontaining a high concentration of sucrose (5%) in order tocounter-select the strains which, after a second homologousrecombination event, have exchanged the chromosomal region of the dcmgene with the homologous fragment cloned into the plasmid. The deletionintroduced into the selected clone (GT106) was verified by PCR with thepair of primers OldcmAF and OldcmBR, generating a fragment smaller insize than that obtained with the parental strain, and by a PCR with theprimer OldcmBR and a primer positioned outside the exchanged region(OldcmCF TTTTGCGGCCGCGTTGCGGTATTACCCTTGTC; SEQ ID No.315). The dcm⁻genotype of the GT106 strain was confirmed by introducing into saidstrain and into GT106, a plasmid containing a restriction site for theSexAI enzyme, which is subject to dcm methylation. The plasmid purifiedfrom GT106 is cleaved by SexAI, whereas it is resistant to the enzymewhen it is purified from GT97.

[0080] The latter strain named GT106 exhibits the same growthcharacteristics as the parental strain GT97 and, as expected, nonegative modification of the amount of R6K gamma plasmid DNAs wasobserved, only the quality of the DNAs, assessed by the absence ofmethylation of the cytosines of the dcm sites, was improved. The GT106strain will be available from the company Invivogen from the day onwhich this patent application is filed.

EXAMPLE 11 Production of the Neo Gene for Resistance to Neomycin, Devoidof CpG

[0081] The Neo ΔCpG gene, the sequence of which is given in FIG. 18(position 3 to 797 of the DNA sequence given in FIG. 18=SEQ ID No. 316;protein sequence ═SEQ ID No. 317), was synthesized from an assembly ofoverlapping oligonucleotides (20-40 pb in size), the sequences of whichare given in FIG. 19. The assembly method is carried out in three steps;the first step consists of phosphorylation of the oligonucleotides ofthe coding strand, in a second step, all the oligonucleotides of bothstrands are combined by hybridization and ligation and, in the finalstep, the gene is amplified by PCR.

[0082] The 20 oligonucleotides of SEQ ID No. 319 to SEQ ID No. 338 (FIG.19) corresponding to the coding strand are phosphorylated according tothe following procedure: 1 μl of each one of the oligonucleotides, takenup in water at 250 μM, are mixed in a microtube containing 50 μl ofwater, so as to bring the final solution to a concentration of 100picomol per microliter. 5 μl of this solution are then mixed with 10 μlof 10-times concentrated polynucleotide kinase buffer, 0.4 μl of a 50 mMATP solution, 85 μl of water and 1 μl of the enzyme (at 10 μ/μl), andthe entire mixture is incubated for 4 hours at 37° C. and then 5 minutesat 95° C. (solution A).

[0083] A solution of the oligonucleotides of the noncoding strand ismade up by mixing 1 μl of each oligonucleotide (SEQ ID No. 339 to SEQ IDNo. 360; FIG. 19) and 1 μl of the oligonucleotide SEQ ID No. 318 (FIG.19), to which solution 160 μl of water are added in order to obtain afinal solution at 54 picomol per μl (solution B).

[0084] The assembly of the gene is carried out first by mixing 10 μl ofsolution A, 1 μl of solution B, 6 μl of a 100 mM KCl solution, 3 μl of a0.5% solution of the surfactant NP-40, 4 μl of a 50 mM MgCl₂ solution, 3μl of a 10 mM ATP solution and 7.5 μl of Pfu ligase (30 units), and themixture is then heated in a programmable thermocycler for 3 minutes at95° C. and then 3 minutes at 80° C., before undergoing 3 cycles of oneminute at 95° C., followed by a change from 95° C. to 70° C. in 1minute, then a change from 70° C. to 55° C. in 1 hour and, finally, 2hours at 55° C. The mixture of the assembled oligonucleotides is thenamplified with the primers NO1 and NO₂₂. The amplification product ispurified on a Promega column, digested with the BspHI and NheIrestriction enzymes and cloned into the plasmid pMOD2LacZ(wt) linearizedwith BspHI and NheI. The sequences of the plasmid DNA of 2kanamycin-resistant clones, which appeared after transformation of theE. coli strain GT100 (available from Invivogen) with the mixture of theligation between the vector fragment and the PCR fragment, were found tobe in accordance with the sequence given in FIG. 18. This syntheticgene, placed under the control of the bacterial EM7 promoter (vectorpMOD2Neo ΔCpG), confers a resistance to kanamycin identical to thatprovided by the same vector containing the native neo gene with therecipient E. coli strain GT100. The BspHI-NheI neo fragment of theplasmid pMOD2Neo ΔCpG was then introduced into the plasmid pSh ΔCpG ofFIG. 16, linearized with NcoI-NheI, to give, after ligation andtransformation in E. coli, the plasmid pNeoΔCpG.

1 360 1 396 DNA Artificial Sequence CpG-Free Sh ble 1 cc atg gcc aag ttgacc agt gct gtc cca gtg ctc aca gcc agg gat 47 Met Ala Lys Leu Thr SerAla Val Pro Val Leu Thr Ala Arg Asp 1 5 10 15 gtg gct gga gct gtt gagttc tgg act gac agg ttg ggg ttc tcc aga 95 Val Ala Gly Ala Val Glu PheTrp Thr Asp Arg Leu Gly Phe Ser Arg 20 25 30 gat ttt gtg gag gat gac tttgca ggt gtg gtc aga gat gat gtc acc 143 Asp Phe Val Glu Asp Asp Phe AlaGly Val Val Arg Asp Asp Val Thr 35 40 45 ctg ttc atc tca gca gtc cag gaccag gtg gtg cct gac aac acc ctg 191 Leu Phe Ile Ser Ala Val Gln Asp GlnVal Val Pro Asp Asn Thr Leu 50 55 60 gct tgg gtg tgg gtg aga gga ctg gatgag ctg tat gct gag tgg agt 239 Ala Trp Val Trp Val Arg Gly Leu Asp GluLeu Tyr Ala Glu Trp Ser 65 70 75 gag gtg gtc tcc acc aac ttc agg gat gccagt ggc cct gcc atg aca 287 Glu Val Val Ser Thr Asn Phe Arg Asp Ala SerGly Pro Ala Met Thr 80 85 90 95 gag att gga gag cag ccc tgg ggg aga gagttt gcc ctg aga gac cca 335 Glu Ile Gly Glu Gln Pro Trp Gly Arg Glu PheAla Leu Arg Asp Pro 100 105 110 gca ggc aac tgt gtg cac ttt gtg gca gaggag cag gac tgaggataag 384 Ala Gly Asn Cys Val His Phe Val Ala Glu GluGln Asp 115 120 aattcagcta gc 396 2 124 PRT Artificial Sequence CpG-FreeSh ble 2 Met Ala Lys Leu Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val1 5 10 15 Ala Gly Ala Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser ArgAsp 20 25 30 Phe Val Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val ThrLeu 35 40 45 Phe Ile Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr LeuAla 50 55 60 Trp Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp SerGlu 65 70 75 80 Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala MetThr Glu 85 90 95 Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg AspPro Ala 100 105 110 Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115120 3 1040 DNA Artificial Sequence CpG-Free Hph 3 tc atg aag aaa cct gaactg aca gca act tct gtt gag aag ttt ctc 47 Met Lys Lys Pro Glu Leu ThrAla Thr Ser Val Glu Lys Phe Leu 1 5 10 15 att gaa aaa ttt gat tct gtttct gat ctc atg cag ctg tct gaa ggt 95 Ile Glu Lys Phe Asp Ser Val SerAsp Leu Met Gln Leu Ser Glu Gly 20 25 30 gaa gaa agc aga gcc ttt tct tttgat gtt gga gga aga ggt tat gtt 143 Glu Glu Ser Arg Ala Phe Ser Phe AspVal Gly Gly Arg Gly Tyr Val 35 40 45 ctg agg gtc aat tct tgt gct gat ggtttt tac aaa gac aga tat gtt 191 Leu Arg Val Asn Ser Cys Ala Asp Gly PheTyr Lys Asp Arg Tyr Val 50 55 60 tac aga cac ttt gcc tct gct gct ctg ccaatt cca gaa gtt ctg gac 239 Tyr Arg His Phe Ala Ser Ala Ala Leu Pro IlePro Glu Val Leu Asp 65 70 75 att gga gaa ttt tct gaa tct ctc acc tac tgcatc agc aga aga gca 287 Ile Gly Glu Phe Ser Glu Ser Leu Thr Tyr Cys IleSer Arg Arg Ala 80 85 90 95 caa gga gtc act ctc cag gat ctc cct gaa actgag ctg cca gct gtt 335 Gln Gly Val Thr Leu Gln Asp Leu Pro Glu Thr GluLeu Pro Ala Val 100 105 110 ctg caa cct gtt gct gaa gca atg gat gcc attgca gca gct gat ctg 383 Leu Gln Pro Val Ala Glu Ala Met Asp Ala Ile AlaAla Ala Asp Leu 115 120 125 agc caa acc tct gga ttt ggt cct ttt ggt ccccaa ggc att ggt cag 431 Ser Gln Thr Ser Gly Phe Gly Pro Phe Gly Pro GlnGly Ile Gly Gln 130 135 140 tac acc act tgg agg gat ttc att tgt gcc attgct gat cct cat gtc 479 Tyr Thr Thr Trp Arg Asp Phe Ile Cys Ala Ile AlaAsp Pro His Val 145 150 155 tat cac tgg cag act gtg atg gat gac aca gtttct gct tct gtt gct 527 Tyr His Trp Gln Thr Val Met Asp Asp Thr Val SerAla Ser Val Ala 160 165 170 175 cag gca ctg gat gaa ctc atg ctg tgg gcagaa gat tgt cct gaa gtc 575 Gln Ala Leu Asp Glu Leu Met Leu Trp Ala GluAsp Cys Pro Glu Val 180 185 190 aga cac ctg gtc cat gct gat ttt gga agcaac aat gtt ctg aca gac 623 Arg His Leu Val His Ala Asp Phe Gly Ser AsnAsn Val Leu Thr Asp 195 200 205 aat ggc aga atc act gca gtc att gac tggtct gaa gcc atg ttt gga 671 Asn Gly Arg Ile Thr Ala Val Ile Asp Trp SerGlu Ala Met Phe Gly 210 215 220 gat tct caa tat gag gtt gcc aac att tttttt tgg aga cct tgg ctg 719 Asp Ser Gln Tyr Glu Val Ala Asn Ile Phe PheTrp Arg Pro Trp Leu 225 230 235 gct tgc atg gaa caa caa aca aga tat tttgaa aga aga cac cca gaa 767 Ala Cys Met Glu Gln Gln Thr Arg Tyr Phe GluArg Arg His Pro Glu 240 245 250 255 ctg gct ggt tcc ccc aga ctg aga gcctac atg ctc aga att ggc ctg 815 Leu Ala Gly Ser Pro Arg Leu Arg Ala TyrMet Leu Arg Ile Gly Leu 260 265 270 gac caa ctg tat caa tct ctg gtt gatgga aac ttt gat gat gct gct 863 Asp Gln Leu Tyr Gln Ser Leu Val Asp GlyAsn Phe Asp Asp Ala Ala 275 280 285 tgg gca caa gga aga tgt gat gcc attgtg agg tct ggt gct gga act 911 Trp Ala Gln Gly Arg Cys Asp Ala Ile ValArg Ser Gly Ala Gly Thr 290 295 300 gtt gga aga act caa att gca aga aggtct gct gct gtt tgg act gat 959 Val Gly Arg Thr Gln Ile Ala Arg Arg SerAla Ala Val Trp Thr Asp 305 310 315 gga tgt gtt gaa gtt ctg gct gac tctgga aac agg aga ccc tcc aca 1007 Gly Cys Val Glu Val Leu Ala Asp Ser GlyAsn Arg Arg Pro Ser Thr 320 325 330 335 aga ccc aga gcc aag gaatgaatattag ctagc 1040 Arg Pro Arg Ala Lys Glu 340 4 341 PRT ArtificialSequence CpG-Free Hph 4 Met Lys Lys Pro Glu Leu Thr Ala Thr Ser Val GluLys Phe Leu Ile 1 5 10 15 Glu Lys Phe Asp Ser Val Ser Asp Leu Met GlnLeu Ser Glu Gly Glu 20 25 30 Glu Ser Arg Ala Phe Ser Phe Asp Val Gly GlyArg Gly Tyr Val Leu 35 40 45 Arg Val Asn Ser Cys Ala Asp Gly Phe Tyr LysAsp Arg Tyr Val Tyr 50 55 60 Arg His Phe Ala Ser Ala Ala Leu Pro Ile ProGlu Val Leu Asp Ile 65 70 75 80 Gly Glu Phe Ser Glu Ser Leu Thr Tyr CysIle Ser Arg Arg Ala Gln 85 90 95 Gly Val Thr Leu Gln Asp Leu Pro Glu ThrGlu Leu Pro Ala Val Leu 100 105 110 Gln Pro Val Ala Glu Ala Met Asp AlaIle Ala Ala Ala Asp Leu Ser 115 120 125 Gln Thr Ser Gly Phe Gly Pro PheGly Pro Gln Gly Ile Gly Gln Tyr 130 135 140 Thr Thr Trp Arg Asp Phe IleCys Ala Ile Ala Asp Pro His Val Tyr 145 150 155 160 His Trp Gln Thr ValMet Asp Asp Thr Val Ser Ala Ser Val Ala Gln 165 170 175 Ala Leu Asp GluLeu Met Leu Trp Ala Glu Asp Cys Pro Glu Val Arg 180 185 190 His Leu ValHis Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn 195 200 205 Gly ArgIle Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe Gly Asp 210 215 220 SerGln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg Pro Trp Leu Ala 225 230 235240 Cys Met Glu Gln Gln Thr Arg Tyr Phe Glu Arg Arg His Pro Glu Leu 245250 255 Ala Gly Ser Pro Arg Leu Arg Ala Tyr Met Leu Arg Ile Gly Leu Asp260 265 270 Gln Leu Tyr Gln Ser Leu Val Asp Gly Asn Phe Asp Asp Ala AlaTrp 275 280 285 Ala Gln Gly Arg Cys Asp Ala Ile Val Arg Ser Gly Ala GlyThr Val 290 295 300 Gly Arg Thr Gln Ile Ala Arg Arg Ser Ala Ala Val TrpThr Asp Gly 305 310 315 320 Cys Val Glu Val Leu Ala Asp Ser Gly Asn ArgArg Pro Ser Thr Arg 325 330 335 Pro Arg Ala Lys Glu 340 5 442 DNAArtificial Sequence CpG-Free Bsr 5 tc atg aag acc ttc aac atc tct cagcag gat ctg gag ctg gtg gag 47 Met Lys Thr Phe Asn Ile Ser Gln Gln AspLeu Glu Leu Val Glu 1 5 10 15 gtc gcc act gag aag atc acc atg ctc tatgag gac aac aag cac cat 95 Val Ala Thr Glu Lys Ile Thr Met Leu Tyr GluAsp Asn Lys His His 20 25 30 gtc ggg gcg gcc atc agg acc aag act ggg gagatc atc tct gct gtc 143 Val Gly Ala Ala Ile Arg Thr Lys Thr Gly Glu IleIle Ser Ala Val 35 40 45 cac att gag gcc tac att ggc agg gtc act gtc tgtgct gaa gcc att 191 His Ile Glu Ala Tyr Ile Gly Arg Val Thr Val Cys AlaGlu Ala Ile 50 55 60 gcc att ggg tct gct gtg agc aac ggg cag aag gac tttgac acc att 239 Ala Ile Gly Ser Ala Val Ser Asn Gly Gln Lys Asp Phe AspThr Ile 65 70 75 gtg gct gtc agg cac ccc tac tct gat gag gtg gac aga tccatc agg 287 Val Ala Val Arg His Pro Tyr Ser Asp Glu Val Asp Arg Ser IleArg 80 85 90 95 gtg gtc agc ccc tgt ggc atg tgc aga gag ctc atc tct gactat gct 335 Val Val Ser Pro Cys Gly Met Cys Arg Glu Leu Ile Ser Asp TyrAla 100 105 110 cct gac tgc ttt gtg ctc att gag atg aat ggc aag ctg gtcaaa acc 383 Pro Asp Cys Phe Val Leu Ile Glu Met Asn Gly Lys Leu Val LysThr 115 120 125 acc att gag gaa ctc atc ccc ctc aag tac acc agg aactaaacctgaa 432 Thr Ile Glu Glu Leu Ile Pro Leu Lys Tyr Thr Arg Asn 130135 140 ttcagctagc 442 6 140 PRT Artificial Sequence CpG-Free Bsr 6 MetLys Thr Phe Asn Ile Ser Gln Gln Asp Leu Glu Leu Val Glu Val 1 5 10 15Ala Thr Glu Lys Ile Thr Met Leu Tyr Glu Asp Asn Lys His His Val 20 25 30Gly Ala Ala Ile Arg Thr Lys Thr Gly Glu Ile Ile Ser Ala Val His 35 40 45Ile Glu Ala Tyr Ile Gly Arg Val Thr Val Cys Ala Glu Ala Ile Ala 50 55 60Ile Gly Ser Ala Val Ser Asn Gly Gln Lys Asp Phe Asp Thr Ile Val 65 70 7580 Ala Val Arg His Pro Tyr Ser Asp Glu Val Asp Arg Ser Ile Arg Val 85 9095 Val Ser Pro Cys Gly Met Cys Arg Glu Leu Ile Ser Asp Tyr Ala Pro 100105 110 Asp Cys Phe Val Leu Ile Glu Met Asn Gly Lys Leu Val Lys Thr Thr115 120 125 Ile Glu Glu Leu Ile Pro Leu Lys Tyr Thr Arg Asn 130 135 1407 614 DNA Artificial Sequence CpG-Free Pac 7 tc atg act gag tac aaa cccaca gtg agg ctg gca acc aga gat gat 47 Met Thr Glu Tyr Lys Pro Thr ValArg Leu Ala Thr Arg Asp Asp 1 5 10 15 gtt cca aga gct gtg aga aca ctggct gct gct ttt gca gac tac cct 95 Val Pro Arg Ala Val Arg Thr Leu AlaAla Ala Phe Ala Asp Tyr Pro 20 25 30 gca aca agg cac aca gtt gac cct gacagg cac att gag agg gtg aca 143 Ala Thr Arg His Thr Val Asp Pro Asp ArgHis Ile Glu Arg Val Thr 35 40 45 gaa ctg caa gaa ctc ttc ctc acc aga gtggga ctg gac att gga aaa 191 Glu Leu Gln Glu Leu Phe Leu Thr Arg Val GlyLeu Asp Ile Gly Lys 50 55 60 gtt tgg gtt gca gat gat gga gct gct gtt gcagtt tgg aca aca cct 239 Val Trp Val Ala Asp Asp Gly Ala Ala Val Ala ValTrp Thr Thr Pro 65 70 75 gag tct gtt gaa gct ggt gct gtt ttt gct gaa attgga cca aga atg 287 Glu Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile GlyPro Arg Met 80 85 90 95 gct gag ctc tct gga agc agg ctg gca gca caa caacaa atg gaa ggt 335 Ala Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln GlnMet Glu Gly 100 105 110 ctg ctg gca cca cac agg cca aaa gag cca gct tggttt ctg gca act 383 Leu Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp PheLeu Ala Thr 115 120 125 gtt gga gtg agc cct gac cac cag gga aag ggt ctggga tct gct gtt 431 Val Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu GlySer Ala Val 130 135 140 gtt ctg cct gga gtt gaa gct gct gaa agg gct ggagtt cct gcc ttt 479 Val Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly ValPro Ala Phe 145 150 155 ctg gaa act tct gct ccc aga aac ctg cct ttt tatgaa aga ctg gga 527 Leu Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr GluArg Leu Gly 160 165 170 175 ttc act gtg aca gct gat gtt gag gtt cca gaaggc cca aga act tgg 575 Phe Thr Val Thr Ala Asp Val Glu Val Pro Glu GlyPro Arg Thr Trp 180 185 190 tgc atg aca agg aag cct gga gct taaacctgagctagc 614 Cys Met Thr Arg Lys Pro Gly Ala 195 8 199 PRT ArtificialSequence CpG-Free Pac 8 Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala ThrArg Asp Asp Val 1 5 10 15 Pro Arg Ala Val Arg Thr Leu Ala Ala Ala PheAla Asp Tyr Pro Ala 20 25 30 Thr Arg His Thr Val Asp Pro Asp Arg His IleGlu Arg Val Thr Glu 35 40 45 Leu Gln Glu Leu Phe Leu Thr Arg Val Gly LeuAsp Ile Gly Lys Val 50 55 60 Trp Val Ala Asp Asp Gly Ala Ala Val Ala ValTrp Thr Thr Pro Glu 65 70 75 80 Ser Val Glu Ala Gly Ala Val Phe Ala GluIle Gly Pro Arg Met Ala 85 90 95 Glu Leu Ser Gly Ser Arg Leu Ala Ala GlnGln Gln Met Glu Gly Leu 100 105 110 Leu Ala Pro His Arg Pro Lys Glu ProAla Trp Phe Leu Ala Thr Val 115 120 125 Gly Val Ser Pro Asp His Gln GlyLys Gly Leu Gly Ser Ala Val Val 130 135 140 Leu Pro Gly Val Glu Ala AlaGlu Arg Ala Gly Val Pro Ala Phe Leu 145 150 155 160 Glu Thr Ser Ala ProArg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe 165 170 175 Thr Val Thr AlaAsp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys 180 185 190 Met Thr ArgLys Pro Gly Ala 195 9 3071 DNA Artificial Sequence CpG-Free LacZ 9 ccatg gac cct gtt gtg ctg caa agg aga gac tgg gag aac cct gga 47 Met AspPro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly 1 5 10 15 gtg acccag ctc aac aga ctg gct gcc cac cct ccc ttt gcc tct tgg 95 Val Thr GlnLeu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Trp 20 25 30 agg aac tctgag gaa gcc agg aca gac agg ccc agc cag cag ctc agg 143 Arg Asn Ser GluGlu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg 35 40 45 tct ctc aat ggagag tgg agg ttt gcc tgg ttc cct gcc cct gaa gct 191 Ser Leu Asn Gly GluTrp Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala 50 55 60 gtg cct gag tct tggctg gag tgt gac ctc cca gag gct gac act gtt 239 Val Pro Glu Ser Trp LeuGlu Cys Asp Leu Pro Glu Ala Asp Thr Val 65 70 75 gtg gtg ccc agc aac tggcag atg cat ggc tat gat gcc ccc atc tac 287 Val Val Pro Ser Asn Trp GlnMet His Gly Tyr Asp Ala Pro Ile Tyr 80 85 90 95 acc aat gtc acc tac cccatc act gtg aac ccc cct ttt gtg ccc act 335 Thr Asn Val Thr Tyr Pro IleThr Val Asn Pro Pro Phe Val Pro Thr 100 105 110 gag aac ccc act ggc tgctac agc ctg acc ttc aat gtt gat gag agc 383 Glu Asn Pro Thr Gly Cys TyrSer Leu Thr Phe Asn Val Asp Glu Ser 115 120 125 tgg ctg caa gaa ggc cagacc agg atc atc ttt gat gga gtc aac tct 431 Trp Leu Gln Glu Gly Gln ThrArg Ile Ile Phe Asp Gly Val Asn Ser 130 135 140 gcc ttc cac ctc tgg tgcaat ggc agg tgg gtt ggc tat ggc caa gac 479 Ala Phe His Leu Trp Cys AsnGly Arg Trp Val Gly Tyr Gly Gln Asp 145 150 155 agc agg ctg ccc tct gagttt gac ctc tct gcc ttc ctc aga gct gga 527 Ser Arg Leu Pro Ser Glu PheAsp Leu Ser Ala Phe Leu Arg Ala Gly 160 165 170 175 gag aac agg ctg gctgtc atg gtg ctc agg tgg tct gat ggc agc tac 575 Glu Asn Arg Leu Ala ValMet Val Leu Arg Trp Ser Asp Gly Ser Tyr 180 185 190 ctg gaa gac caa gacatg tgg agg atg tct ggc atc ttc agg gat gtg 623 Leu Glu Asp Gln Asp MetTrp Arg Met Ser Gly Ile Phe Arg Asp Val 195 200 205 agc ctg ctg cac aagccc acc acc cag att tct gac ttc cat gtt gcc 671 Ser Leu Leu His Lys ProThr Thr Gln Ile Ser Asp Phe His Val Ala 210 215 220 acc agg ttc aat gatgac ttc agc aga gct gtg ctg gag gct gag gtg 719 Thr Arg Phe Asn Asp AspPhe Ser Arg Ala Val Leu Glu Ala Glu Val 225 230 235 cag atg tgt gga gaactc aga gac tac ctg aga gtc aca gtg agc ctc 767 Gln Met Cys Gly Glu LeuArg Asp Tyr Leu Arg Val Thr Val Ser Leu 240 245 250 255 tgg caa ggt gagacc cag gtg gcc tct ggc aca gcc ccc ttt gga gga 815 Trp Gln Gly Glu ThrGln Val Ala Ser Gly Thr Ala Pro Phe Gly Gly 260 265 270 gag atc att gatgag aga gga ggc tat gct gac aga gtc acc ctg agg 863 Glu Ile Ile Asp GluArg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg 275 280 285 ctc aat gtg gagaac ccc aag ctg tgg tct gct gag atc ccc aac ctc 911 Leu Asn Val Glu AsnPro Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu 290 295 300 tac agg gct gttgtg gag ctg cac act gct gat ggc acc ctg att gaa 959 Tyr Arg Ala Val ValGlu Leu His Thr Ala Asp Gly Thr Leu Ile Glu 305 310 315 gct gaa gcc tgtgat gtt gga ttc aga gaa gtc agg att gag aat ggc 1007 Ala Glu Ala Cys AspVal Gly Phe Arg Glu Val Arg Ile Glu Asn Gly 320 325 330 335 ctg ctg ctgctc aat ggc aag cct ctg ctc atc agg gga gtc aac agg 1055 Leu Leu Leu LeuAsn Gly Lys Pro Leu Leu Ile Arg Gly Val Asn Arg 340 345 350 cat gag caccac cct ctg cat gga caa gtg atg gat gaa cag aca atg 1103 His Glu His HisPro Leu His Gly Gln Val Met Asp Glu Gln Thr Met 355 360 365 gtg caa gatatc ctg cta atg aag cag aac aac ttc aat gct gtc agg 1151 Val Gln Asp IleLeu Leu Met Lys Gln Asn Asn Phe Asn Ala Val Arg 370 375 380 tgc tct cactac ccc aac cac cct ctc tgg tac acc ctg tgt gac agg 1199 Cys Ser His TyrPro Asn His Pro Leu Trp Tyr Thr Leu Cys Asp Arg 385 390 395 tat ggc ctgtat gtt gtt gat gaa gcc aac att gag aca cat ggc atg 1247 Tyr Gly Leu TyrVal Val Asp Glu Ala Asn Ile Glu Thr His Gly Met 400 405 410 415 gtg cccatg aac agg ctc aca gat gac ccc agg tgg ctg cct gcc atg 1295 Val Pro MetAsn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro Ala Met 420 425 430 tct gagaga gtg acc agg atg gtg cag aga gac agg aac cac ccc tct 1343 Ser Glu ArgVal Thr Arg Met Val Gln Arg Asp Arg Asn His Pro Ser 435 440 445 gtg atcatc tgg tct ctg ggc aat gag tct gga cat gga gcc aac cat 1391 Val Ile IleTrp Ser Leu Gly Asn Glu Ser Gly His Gly Ala Asn His 450 455 460 gat gctctc tac agg tgg atc aag tct gtt gac ccc agc aga cct gtg 1439 Asp Ala LeuTyr Arg Trp Ile Lys Ser Val Asp Pro Ser Arg Pro Val 465 470 475 cag tatgaa gga ggt gga gca gac acc aca gcc aca gac atc atc tgc 1487 Gln Tyr GluGly Gly Gly Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys 480 485 490 495 cccatg tat gcc agg gtt gat gag gac cag ccc ttc cct gct gtg ccc 1535 Pro MetTyr Ala Arg Val Asp Glu Asp Gln Pro Phe Pro Ala Val Pro 500 505 510 aagtgg agc atc aag aag tgg ctc tct ctg cct gga gag acc aga cct 1583 Lys TrpSer Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro 515 520 525 ctgatc ctg tgt gaa tat gca cat gca atg ggc aac tct ctg gga ggc 1631 Leu IleLeu Cys Glu Tyr Ala His Ala Met Gly Asn Ser Leu Gly Gly 530 535 540 tttgcc aag tac tgg caa gcc ttc aga cag tac ccc agg ctg caa gga 1679 Phe AlaLys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly 545 550 555 ggattt gtg tgg gac tgg gtg gac caa tct ctc atc aag tat gat gag 1727 Gly PheVal Trp Asp Trp Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu 560 565 570 575aat ggc aac ccc tgg tct gcc tat gga gga gac ttt ggt gac acc ccc 1775 AsnGly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro 580 585 590aat gac agg cag ttc tgc atg aat ggc ctg gtc ttt gca gac agg acc 1823 AsnAsp Arg Gln Phe Cys Met Asn Gly Leu Val Phe Ala Asp Arg Thr 595 600 605cct cac cct gcc ctc aca gag gcc aag cac cag caa cag ttc ttc cag 1871 ProHis Pro Ala Leu Thr Glu Ala Lys His Gln Gln Gln Phe Phe Gln 610 615 620ttc agg ctg tct gga cag acc att gag gtg aca tct gag tac ctc ttc 1919 PheArg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe 625 630 635agg cac tct gac aat gag ctc ctg cac tgg atg gtg gcc ctg gat ggc 1967 ArgHis Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu Asp Gly 640 645 650655 aag cct ctg gct tct ggt gag gtg cct ctg gat gtg gcc cct caa gga 2015Lys Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro Gln Gly 660 665670 aag cag ctg att gaa ctg cct gag ctg cct cag cca gag tct gct gga 2063Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly 675 680685 caa ctg tgg cta aca gtg agg gtg gtt cag ccc aat gca aca gct tgg 2111Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn Ala Thr Ala Trp 690 695700 tct gag gca ggc cac atc tct gca tgg cag cag tgg agg ctg gct gag 2159Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu 705 710715 aac ctc tct gtg acc ctg cct gct gcc tct cat gcc atc cct cac ctg 2207Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala Ile Pro His Leu 720 725730 735 aca aca tct gaa atg gac ttc tgc att gag ctg ggc aac aag aga tgg2255 Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp 740745 750 cag ttc aac agg cag tct ggc ttc ctg tct cag atg tgg att gga gac2303 Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp 755760 765 aag aag cag ctc ctc acc cct ctc agg gac caa ttc acc agg gct cct2351 Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro 770775 780 ctg gac aat gac att gga gtg tct gag gcc acc agg att gac cca aat2399 Leu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro Asn 785790 795 gct tgg gtg gag agg tgg aag gct gct gga cac tac cag gct gag gct2447 Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr Gln Ala Glu Ala 800805 810 815 gcc ctg ctc cag tgc aca gca gac acc ctg gct gat gct gtt ctgatc 2495 Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp Ala Val Leu Ile820 825 830 acc aca gcc cat gct tgg cag cac caa ggc aag acc ctg ttc atcagc 2543 Thr Thr Ala His Ala Trp Gln His Gln Gly Lys Thr Leu Phe Ile Ser835 840 845 aga aag acc tac agg att gat ggc tct gga cag atg gca atc acagtg 2591 Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met Ala Ile Thr Val850 855 860 gat gtg gag gtt gcc tct gac aca cct cac cct gca agg att ggcctg 2639 Asp Val Glu Val Ala Ser Asp Thr Pro His Pro Ala Arg Ile Gly Leu865 870 875 aac tgt caa ctg gca cag gtg gct gag agg gtg aac tgg ctg ggctta 2687 Asn Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn Trp Leu Gly Leu880 885 890 895 ggc cct cag gag aac tac cct gac agg ctg aca gct gcc tgcttt gac 2735 Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala Ala Cys PheAsp 900 905 910 agg tgg gac ctg cct ctg tct gac atg tac acc cct tat gtgttc cct 2783 Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val PhePro 915 920 925 tct gag aat ggc ctg agg tgt ggc acc agg gag ctg aac tatggt cct 2831 Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr GlyPro 930 935 940 cac cag tgg agg gga gac ttc cag ttc aac atc tcc agg tactct cag 2879 His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr SerGln 945 950 955 caa cag ctc atg gaa acc tct cac agg cac ctg ctc cat gcagag gag 2927 Gln Gln Leu Met Glu Thr Ser His Arg His Leu Leu His Ala GluGlu 960 965 970 975 gga acc tgg ctg aac att gat ggc ttc cac atg ggc attgga gga gat 2975 Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly Ile GlyGly Asp 980 985 990 gac tct tgg tct cct tct gtg tct gct gag ttc cag ttatct gct ggc 3023 Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln Leu SerAla Gly 995 1000 1005 agg tac cac tat cag ctg gtg tgg tgc cag aagtaaacctgag ctagc 3071 Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys 10101015 10 1018 PRT Artificial Sequence CpG-Free LacZ 10 Met Asp Pro ValVal Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr Gln LeuAsn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Trp Arg 20 25 30 Asn Ser GluGlu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser 35 40 45 Leu Asn GlyGlu Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val 50 55 60 Pro Glu SerTrp Leu Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val 65 70 75 80 Val ProSer Asn Trp Gln Met His Gly Tyr Asp Ala Pro Ile Tyr Thr 85 90 95 Asn ValThr Tyr Pro Ile Thr Val Asn Pro Pro Phe Val Pro Thr Glu 100 105 110 AsnPro Thr Gly Cys Tyr Ser Leu Thr Phe Asn Val Asp Glu Ser Trp 115 120 125Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp Gly Val Asn Ser Ala 130 135140 Phe His Leu Trp Cys Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser 145150 155 160 Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe Leu Arg Ala GlyGlu 165 170 175 Asn Arg Leu Ala Val Met Val Leu Arg Trp Ser Asp Gly SerTyr Leu 180 185 190 Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile Phe ArgAsp Val Ser 195 200 205 Leu Leu His Lys Pro Thr Thr Gln Ile Ser Asp PheHis Val Ala Thr 210 215 220 Arg Phe Asn Asp Asp Phe Ser Arg Ala Val LeuGlu Ala Glu Val Gln 225 230 235 240 Met Cys Gly Glu Leu Arg Asp Tyr LeuArg Val Thr Val Ser Leu Trp 245 250 255 Gln Gly Glu Thr Gln Val Ala SerGly Thr Ala Pro Phe Gly Gly Glu 260 265 270 Ile Ile Asp Glu Arg Gly GlyTyr Ala Asp Arg Val Thr Leu Arg Leu 275 280 285 Asn Val Glu Asn Pro LysLeu Trp Ser Ala Glu Ile Pro Asn Leu Tyr 290 295 300 Arg Ala Val Val GluLeu His Thr Ala Asp Gly Thr Leu Ile Glu Ala 305 310 315 320 Glu Ala CysAsp Val Gly Phe Arg Glu Val Arg Ile Glu Asn Gly Leu 325 330 335 Leu LeuLeu Asn Gly Lys Pro Leu Leu Ile Arg Gly Val Asn Arg His 340 345 350 GluHis His Pro Leu His Gly Gln Val Met Asp Glu Gln Thr Met Val 355 360 365Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe Asn Ala Val Arg Cys 370 375380 Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr 385390 395 400 Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu Thr His Gly MetVal 405 410 415 Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro AlaMet Ser 420 425 430 Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg Asn HisPro Ser Val 435 440 445 Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His GlyAla Asn His Asp 450 455 460 Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp ProSer Arg Pro Val Gln 465 470 475 480 Tyr Glu Gly Gly Gly Ala Asp Thr ThrAla Thr Asp Ile Ile Cys Pro 485 490 495 Met Tyr Ala Arg Val Asp Glu AspGln Pro Phe Pro Ala Val Pro Lys 500 505 510 Trp Ser Ile Lys Lys Trp LeuSer Leu Pro Gly Glu Thr Arg Pro Leu 515 520 525 Ile Leu Cys Glu Tyr AlaHis Ala Met Gly Asn Ser Leu Gly Gly Phe 530 535 540 Ala Lys Tyr Trp GlnAla Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly 545 550 555 560 Phe Val TrpAsp Trp Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn 565 570 575 Gly AsnPro Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn 580 585 590 AspArg Gln Phe Cys Met Asn Gly Leu Val Phe Ala Asp Arg Thr Pro 595 600 605His Pro Ala Leu Thr Glu Ala Lys His Gln Gln Gln Phe Phe Gln Phe 610 615620 Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg 625630 635 640 His Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu Asp GlyLys 645 650 655 Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro GlnGly Lys 660 665 670 Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu SerAla Gly Gln 675 680 685 Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn AlaThr Ala Trp Ser 690 695 700 Glu Ala Gly His Ile Ser Ala Trp Gln Gln TrpArg Leu Ala Glu Asn 705 710 715 720 Leu Ser Val Thr Leu Pro Ala Ala SerHis Ala Ile Pro His Leu Thr 725 730 735 Thr Ser Glu Met Asp Phe Cys IleGlu Leu Gly Asn Lys Arg Trp Gln 740 745 750 Phe Asn Arg Gln Ser Gly PheLeu Ser Gln Met Trp Ile Gly Asp Lys 755 760 765 Lys Gln Leu Leu Thr ProLeu Arg Asp Gln Phe Thr Arg Ala Pro Leu 770 775 780 Asp Asn Asp Ile GlyVal Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala 785 790 795 800 Trp Val GluArg Trp Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala 805 810 815 Leu LeuGln Cys Thr Ala Asp Thr Leu Ala Asp Ala Val Leu Ile Thr 820 825 830 ThrAla His Ala Trp Gln His Gln Gly Lys Thr Leu Phe Ile Ser Arg 835 840 845Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met Ala Ile Thr Val Asp 850 855860 Val Glu Val Ala Ser Asp Thr Pro His Pro Ala Arg Ile Gly Leu Asn 865870 875 880 Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn Trp Leu Gly LeuGly 885 890 895 Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala Ala Cys PheAsp Arg 900 905 910 Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr ValPhe Pro Ser 915 920 925 Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu AsnTyr Gly Pro His 930 935 940 Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile SerArg Tyr Ser Gln Gln 945 950 955 960 Gln Leu Met Glu Thr Ser His Arg HisLeu Leu His Ala Glu Glu Gly 965 970 975 Thr Trp Leu Asn Ile Asp Gly PheHis Met Gly Ile Gly Gly Asp Asp 980 985 990 Ser Trp Ser Pro Ser Val SerAla Glu Phe Gln Leu Ser Ala Gly Arg 995 1000 1005 Tyr His Tyr Gln LeuVal Trp Cys Gln Lys 1010 1015 11 66 DNA Artificial Sequence CpG-Freeconstitutive promotor 11 caattaaaca ttggcatagt atatctgcat agtataatacaactcactat aggagggcca 60 ccatgg 66 12 320 DNA Artificial SequenceCpG-Free replication origin 12 gcaggactga ggcttaatta aaccttaaaacctttaaaag ccttatatat tctttttttt 60 cttataaaac ttaaaacctt agaggctatttaagttgctg atttatatta attttattgt 120 tcaaacatga gagcttagta catgaaacatgagagcttag tacattagcc atgagagctt 180 agtacattag ccatgagggt ttagttcattaaacatgaga gcttagtaca ttaaacatga 240 gagcttagta catgaaacat gagagcttagtacatactat caacaggttg aactgctgat 300 cttaattaac ctggagactt 320 13 273DNA Artificial Sequence CpG-Free replication origin 13 ttaattaaccttaaaacctt taaaagcctt atatattctt ttttttctta taaaacttaa 60 aaccttagaggctatttaag ttgctgattt atattaattt tattgttcaa acatgagagc 120 ttagtacatgaaacatgaga gcttagtaca ttagccatga gagcttagta cattagccat 180 gagggtttagttcattaaac atgagagctt agtacattaa acatgagagc ttagtacata 240 ctatcaacaggttgaactgc tgatcttaat taa 273 14 3852 DNA Artificial Sequence SEQ IDNO14 = CpG-Free pSh-LacZ plasmid ; SEQ ID NO15= SEQ ID NO14 CDS from 83to 454 (SEQ ID NO2) + SEQ ID NO14 CDS from 483 to 3536 (SEQ ID NO10) 14ttaattaaaa cagtagttga caattaaaca ttggcatagt atatctgcat agtataatac 60aactcactat aggagggcca cc atg gcc aag ttg acc agt gct gtc cca gtg 112 MetAla Lys Leu Thr Ser Ala Val Pro Val 1 5 10 ctc aca gcc agg gat gtg gctgga gct gtt gag ttc tgg act gac agg 160 Leu Thr Ala Arg Asp Val Ala GlyAla Val Glu Phe Trp Thr Asp Arg 15 20 25 ttg ggg ttc tcc aga gat ttt gtggag gat gac ttt gca ggt gtg gtc 208 Leu Gly Phe Ser Arg Asp Phe Val GluAsp Asp Phe Ala Gly Val Val 30 35 40 aga gat gat gtc acc ctg ttc atc tcagca gtc cag gac cag gtg gtg 256 Arg Asp Asp Val Thr Leu Phe Ile Ser AlaVal Gln Asp Gln Val Val 45 50 55 cct gac aac acc ctg gct tgg gtg tgg gtgaga gga ctg gat gag ctg 304 Pro Asp Asn Thr Leu Ala Trp Val Trp Val ArgGly Leu Asp Glu Leu 60 65 70 tat gct gag tgg agt gag gtg gtc tcc acc aacttc agg gat gcc agt 352 Tyr Ala Glu Trp Ser Glu Val Val Ser Thr Asn PheArg Asp Ala Ser 75 80 85 90 ggc cct gcc atg aca gag att gga gag cag ccctgg ggg aga gag ttt 400 Gly Pro Ala Met Thr Glu Ile Gly Glu Gln Pro TrpGly Arg Glu Phe 95 100 105 gcc ctg aga gac cca gca ggc aac tgt gtg cacttt gtg gca gag gag 448 Ala Leu Arg Asp Pro Ala Gly Asn Cys Val His PheVal Ala Glu Glu 110 115 120 cag gac tgaggataag aattctgagg agaagctc atggac cct gtt gtg ctg 500 Gln Asp Met Asp Pro Val Val Leu 125 130 caa aggaga gac tgg gag aac cct gga gtg acc cag ctc aac aga ctg 548 Gln Arg ArgAsp Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu 135 140 145 gct gcccac cct ccc ttt gcc tct tgg agg aac tct gag gaa gcc agg 596 Ala Ala HisPro Pro Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg 150 155 160 aca gacagg ccc agc cag cag ctc agg tct ctc aat gga gag tgg agg 644 Thr Asp ArgPro Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg 165 170 175 ttt gcctgg ttc cct gcc cct gaa gct gtg cct gag tct tgg ctg gag 692 Phe Ala TrpPhe Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu 180 185 190 tgt gacctc cca gag gct gac act gtt gtg gtg ccc agc aac tgg cag 740 Cys Asp LeuPro Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln 195 200 205 210 atgcat ggc tat gat gcc ccc atc tac acc aat gtc acc tac ccc atc 788 Met HisGly Tyr Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile 215 220 225 actgtg aac ccc cct ttt gtg ccc act gag aac ccc act ggc tgc tac 836 Thr ValAsn Pro Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr 230 235 240 agcctg acc ttc aat gtt gat gag agc tgg ctg caa gaa ggc cag acc 884 Ser LeuThr Phe Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr 245 250 255 aggatc atc ttt gat gga gtc aac tct gcc ttc cac ctc tgg tgc aat 932 Arg IleIle Phe Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn 260 265 270 ggcagg tgg gtt ggc tat ggc caa gac agc agg ctg ccc tct gag ttt 980 Gly ArgTrp Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe 275 280 285 290gac ctc tct gcc ttc ctc aga gct gga gag aac agg ctg gct gtc atg 1028 AspLeu Ser Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met 295 300 305gtg ctc agg tgg tct gat ggc agc tac ctg gaa gac caa gac atg tgg 1076 ValLeu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp 310 315 320agg atg tct ggc atc ttc agg gat gtg agc ctg ctg cac aag ccc acc 1124 ArgMet Ser Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys Pro Thr 325 330 335acc cag att tct gac ttc cat gtt gcc acc agg ttc aat gat gac ttc 1172 ThrGln Ile Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp Asp Phe 340 345 350agc aga gct gtg ctg gag gct gag gtg cag atg tgt gga gaa ctc aga 1220 SerArg Ala Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg 355 360 365370 gac tac ctg aga gtc aca gtg agc ctc tgg caa ggt gag acc cag gtg 1268Asp Tyr Leu Arg Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val 375 380385 gcc tct ggc aca gcc ccc ttt gga gga gag atc att gat gag aga gga 1316Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly 390 395400 ggc tat gct gac aga gtc acc ctg agg ctc aat gtg gag aac ccc aag 1364Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys 405 410415 ctg tgg tct gct gag atc ccc aac ctc tac agg gct gtt gtg gag ctg 1412Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu 420 425430 cac act gct gat ggc acc ctg att gaa gct gaa gcc tgt gat gtt gga 1460His Thr Ala Asp Gly Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly 435 440445 450 ttc aga gaa gtc agg att gag aat ggc ctg ctg ctg ctc aat ggc aag1508 Phe Arg Glu Val Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly Lys 455460 465 cct ctg ctc atc agg gga gtc aac agg cat gag cac cac cct ctg cat1556 Pro Leu Leu Ile Arg Gly Val Asn Arg His Glu His His Pro Leu His 470475 480 gga caa gtg atg gat gaa cag aca atg gtg caa gat atc ctg cta atg1604 Gly Gln Val Met Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met 485490 495 aag cag aac aac ttc aat gct gtc agg tgc tct cac tac ccc aac cac1652 Lys Gln Asn Asn Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn His 500505 510 cct ctc tgg tac acc ctg tgt gac agg tat ggc ctg tat gtt gtt gat1700 Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp 515520 525 530 gaa gcc aac att gag aca cat ggc atg gtg ccc atg aac agg ctcaca 1748 Glu Ala Asn Ile Glu Thr His Gly Met Val Pro Met Asn Arg Leu Thr535 540 545 gat gac ccc agg tgg ctg cct gcc atg tct gag aga gtg acc aggatg 1796 Asp Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met550 555 560 gtg cag aga gac agg aac cac ccc tct gtg atc atc tgg tct ctgggc 1844 Val Gln Arg Asp Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu Gly565 570 575 aat gag tct gga cat gga gcc aac cat gat gct ctc tac agg tggatc 1892 Asn Glu Ser Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg Trp Ile580 585 590 aag tct gtt gac ccc agc aga cct gtg cag tat gaa gga ggt ggagca 1940 Lys Ser Val Asp Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala595 600 605 610 gac acc aca gcc aca gac atc atc tgc ccc atg tat gcc agggtt gat 1988 Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg ValAsp 615 620 625 gag gac cag ccc ttc cct gct gtg ccc aag tgg agc atc aagaag tgg 2036 Glu Asp Gln Pro Phe Pro Ala Val Pro Lys Trp Ser Ile Lys LysTrp 630 635 640 ctc tct ctg cct gga gag acc aga cct ctg atc ctg tgt gaatat gca 2084 Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu TyrAla 645 650 655 cat gca atg ggc aac tct ctg gga ggc ttt gcc aag tac tggcaa gcc 2132 His Ala Met Gly Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp GlnAla 660 665 670 ttc aga cag tac ccc agg ctg caa gga gga ttt gtg tgg gactgg gtg 2180 Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly Phe Val Trp Asp TrpVal 675 680 685 690 gac caa tct ctc atc aag tat gat gag aat ggc aac ccctgg tct gcc 2228 Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn Gly Asn Pro TrpSer Ala 695 700 705 tat gga gga gac ttt ggt gac acc ccc aat gac agg cagttc tgc atg 2276 Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn Asp Arg Gln PheCys Met 710 715 720 aat ggc ctg gtc ttt gca gac agg acc cct cac cct gccctc aca gag 2324 Asn Gly Leu Val Phe Ala Asp Arg Thr Pro His Pro Ala LeuThr Glu 725 730 735 gcc aag cac cag caa cag ttc ttc cag ttc agg ctg tctgga cag acc 2372 Ala Lys His Gln Gln Gln Phe Phe Gln Phe Arg Leu Ser GlyGln Thr 740 745 750 att gag gtg aca tct gag tac ctc ttc agg cac tct gacaat gag ctc 2420 Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg His Ser Asp AsnGlu Leu 755 760 765 770 ctg cac tgg atg gtg gcc ctg gat ggc aag cct ctggct tct ggt gag 2468 Leu His Trp Met Val Ala Leu Asp Gly Lys Pro Leu AlaSer Gly Glu 775 780 785 gtg cct ctg gat gtg gcc cct caa gga aag cag ctgatt gaa ctg cct 2516 Val Pro Leu Asp Val Ala Pro Gln Gly Lys Gln Leu IleGlu Leu Pro 790 795 800 gag ctg cct cag cca gag tct gct gga caa ctg tggcta aca gtg agg 2564 Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln Leu Trp LeuThr Val Arg 805 810 815 gtg gtt cag ccc aat gca aca gct tgg tct gag gcaggc cac atc tct 2612 Val Val Gln Pro Asn Ala Thr Ala Trp Ser Glu Ala GlyHis Ile Ser 820 825 830 gca tgg cag cag tgg agg ctg gct gag aac ctc tctgtg acc ctg cct 2660 Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn Leu Ser ValThr Leu Pro 835 840 845 850 gct gcc tct cat gcc atc cct cac ctg aca acatct gaa atg gac ttc 2708 Ala Ala Ser His Ala Ile Pro His Leu Thr Thr SerGlu Met Asp Phe 855 860 865 tgc att gag ctg ggc aac aag aga tgg cag ttcaac agg cag tct ggc 2756 Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln Phe AsnArg Gln Ser Gly 870 875 880 ttc ctg tct cag atg tgg att gga gac aag aagcag ctc ctc acc cct 2804 Phe Leu Ser Gln Met Trp Ile Gly Asp Lys Lys GlnLeu Leu Thr Pro 885 890 895 ctc agg gac caa ttc acc agg gct cct ctg gacaat gac att gga gtg 2852 Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu Asp AsnAsp Ile Gly Val 900 905 910 tct gag gcc acc agg att gac cca aat gct tgggtg gag agg tgg aag 2900 Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala Trp ValGlu Arg Trp Lys 915 920 925 930 gct gct gga cac tac cag gct gag gct gccctg ctc cag tgc aca gca 2948 Ala Ala Gly His Tyr Gln Ala Glu Ala Ala LeuLeu Gln Cys Thr Ala 935 940 945 gac acc ctg gct gat gct gtt ctg atc accaca gcc cat gct tgg cag 2996 Asp Thr Leu Ala Asp Ala Val Leu Ile Thr ThrAla His Ala Trp Gln 950 955 960 cac caa ggc aag acc ctg ttc atc agc agaaag acc tac agg att gat 3044 His Gln Gly Lys Thr Leu Phe Ile Ser Arg LysThr Tyr Arg Ile Asp 965 970 975 ggc tct gga cag atg gca atc aca gtg gatgtg gag gtt gcc tct gac 3092 Gly Ser Gly Gln Met Ala Ile Thr Val Asp ValGlu Val Ala Ser Asp 980 985 990 aca cct cac cct gca agg att ggc ctg aactgt caa ctg gca cag 3137 Thr Pro His Pro Ala Arg Ile Gly Leu Asn Cys GlnLeu Ala Gln 995 1000 1005 gtg gct gag agg gtg aac tgg ctg ggc tta ggccct cag gag aac 3182 Val Ala Glu Arg Val Asn Trp Leu Gly Leu Gly Pro GlnGlu Asn 1010 1015 1020 tac cct gac agg ctg aca gct gcc tgc ttt gac aggtgg gac ctg 3227 Tyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg Trp AspLeu 1025 1030 1035 cct ctg tct gac atg tac acc cct tat gtg ttc cct tctgag aat 3272 Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser Glu Asn1040 1045 1050 ggc ctg agg tgt ggc acc agg gag ctg aac tat ggt cct caccag 3317 Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His Gln1055 1060 1065 tgg agg gga gac ttc cag ttc aac atc tcc agg tac tct cagcaa 3362 Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln Gln1070 1075 1080 cag ctc atg gaa acc tct cac agg cac ctg ctc cat gca gaggag 3407 Gln Leu Met Glu Thr Ser His Arg His Leu Leu His Ala Glu Glu1085 1090 1095 gga acc tgg ctg aac att gat ggc ttc cac atg ggc att ggagga 3452 Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly Ile Gly Gly1100 1105 1110 gat gac tct tgg tct cct tct gtg tct gct gag ttc cag ttatct 3497 Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln Leu Ser1115 1120 1125 gct ggc agg tac cac tat cag ctg gtg tgg tgc cag aagtaagctagct 3546 Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys 11301135 1140 gagtttcaga aaagggggcc tgagtggccc cttttttcaa cttaattaaccttaaaacct 3606 ttaaaagcct tatatattct tttttttctt ataaaactta aaaccttagaggctatttaa 3666 gttgctgatt tatattaatt ttattgttca aacatgagag cttagtacatgaaacatgag 3726 agcttagtac attagccatg agagcttagt acattagcca tgagggtttagttcattaaa 3786 catgagagct tagtacatta aacatgagag cttagtacat actatcaacaggttgaactg 3846 ctgatc 3852 15 1142 PRT Artificial Sequence SEQ ID NO14= CpG-Free pSh-LacZ plasmid ; SEQ ID NO15= SEQ ID NO14 CDS from 83 to454 (SEQ ID NO2) + SEQ ID NO14 CDS from 483 to 3536 (SEQ ID NO10) 15 MetAla Lys Leu Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val 1 5 10 15Ala Gly Ala Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30Phe Val Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45Phe Ile Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55 60Trp Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu 65 70 7580 Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr Glu 85 9095 Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro Ala 100105 110 Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp Met Asp Pro Val115 120 125 Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val Thr Gln LeuAsn 130 135 140 Arg Leu Ala Ala His Pro Pro Phe Ala Ser Trp Arg Asn SerGlu Glu 145 150 155 160 Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg SerLeu Asn Gly Glu 165 170 175 Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu AlaVal Pro Glu Ser Trp 180 185 190 Leu Glu Cys Asp Leu Pro Glu Ala Asp ThrVal Val Val Pro Ser Asn 195 200 205 Trp Gln Met His Gly Tyr Asp Ala ProIle Tyr Thr Asn Val Thr Tyr 210 215 220 Pro Ile Thr Val Asn Pro Pro PheVal Pro Thr Glu Asn Pro Thr Gly 225 230 235 240 Cys Tyr Ser Leu Thr PheAsn Val Asp Glu Ser Trp Leu Gln Glu Gly 245 250 255 Gln Thr Arg Ile IlePhe Asp Gly Val Asn Ser Ala Phe His Leu Trp 260 265 270 Cys Asn Gly ArgTrp Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser 275 280 285 Glu Phe AspLeu Ser Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala 290 295 300 Val MetVal Leu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp 305 310 315 320Met Trp Arg Met Ser Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys 325 330335 Pro Thr Thr Gln Ile Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp 340345 350 Asp Phe Ser Arg Ala Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu355 360 365 Leu Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp Gln Gly GluThr 370 375 380 Gln Val Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu Ile IleAsp Glu 385 390 395 400 Arg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg LeuAsn Val Glu Asn 405 410 415 Pro Lys Leu Trp Ser Ala Glu Ile Pro Asn LeuTyr Arg Ala Val Val 420 425 430 Glu Leu His Thr Ala Asp Gly Thr Leu IleGlu Ala Glu Ala Cys Asp 435 440 445 Val Gly Phe Arg Glu Val Arg Ile GluAsn Gly Leu Leu Leu Leu Asn 450 455 460 Gly Lys Pro Leu Leu Ile Arg GlyVal Asn Arg His Glu His His Pro 465 470 475 480 Leu His Gly Gln Val MetAsp Glu Gln Thr Met Val Gln Asp Ile Leu 485 490 495 Leu Met Lys Gln AsnAsn Phe Asn Ala Val Arg Cys Ser His Tyr Pro 500 505 510 Asn His Pro LeuTrp Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val 515 520 525 Val Asp GluAla Asn Ile Glu Thr His Gly Met Val Pro Met Asn Arg 530 535 540 Leu ThrAsp Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr 545 550 555 560Arg Met Val Gln Arg Asp Arg Asn His Pro Ser Val Ile Ile Trp Ser 565 570575 Leu Gly Asn Glu Ser Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg 580585 590 Trp Ile Lys Ser Val Asp Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly595 600 605 Gly Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro Met Tyr AlaArg 610 615 620 Val Asp Glu Asp Gln Pro Phe Pro Ala Val Pro Lys Trp SerIle Lys 625 630 635 640 Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro LeuIle Leu Cys Glu 645 650 655 Tyr Ala His Ala Met Gly Asn Ser Leu Gly GlyPhe Ala Lys Tyr Trp 660 665 670 Gln Ala Phe Arg Gln Tyr Pro Arg Leu GlnGly Gly Phe Val Trp Asp 675 680 685 Trp Val Asp Gln Ser Leu Ile Lys TyrAsp Glu Asn Gly Asn Pro Trp 690 695 700 Ser Ala Tyr Gly Gly Asp Phe GlyAsp Thr Pro Asn Asp Arg Gln Phe 705 710 715 720 Cys Met Asn Gly Leu ValPhe Ala Asp Arg Thr Pro His Pro Ala Leu 725 730 735 Thr Glu Ala Lys HisGln Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly 740 745 750 Gln Thr Ile GluVal Thr Ser Glu Tyr Leu Phe Arg His Ser Asp Asn 755 760 765 Glu Leu LeuHis Trp Met Val Ala Leu Asp Gly Lys Pro Leu Ala Ser 770 775 780 Gly GluVal Pro Leu Asp Val Ala Pro Gln Gly Lys Gln Leu Ile Glu 785 790 795 800Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr 805 810815 Val Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser Glu Ala Gly His 820825 830 Ile Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn Leu Ser Val Thr835 840 845 Leu Pro Ala Ala Ser His Ala Ile Pro His Leu Thr Thr Ser GluMet 850 855 860 Asp Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln Phe AsnArg Gln 865 870 875 880 Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp LysLys Gln Leu Leu 885 890 895 Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala ProLeu Asp Asn Asp Ile 900 905 910 Gly Val Ser Glu Ala Thr Arg Ile Asp ProAsn Ala Trp Val Glu Arg 915 920 925 Trp Lys Ala Ala Gly His Tyr Gln AlaGlu Ala Ala Leu Leu Gln Cys 930 935 940 Thr Ala Asp Thr Leu Ala Asp AlaVal Leu Ile Thr Thr Ala His Ala 945 950 955 960 Trp Gln His Gln Gly LysThr Leu Phe Ile Ser Arg Lys Thr Tyr Arg 965 970 975 Ile Asp Gly Ser GlyGln Met Ala Ile Thr Val Asp Val Glu Val Ala 980 985 990 Ser Asp Thr ProHis Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala 995 1000 1005 Gln ValAla Glu Arg Val Asn Trp Leu Gly Leu Gly Pro Gln Glu 1010 1015 1020 AsnTyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg Trp Asp 1025 1030 1035Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser Glu 1040 10451050 Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His 10551060 1065 Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln1070 1075 1080 Gln Gln Leu Met Glu Thr Ser His Arg His Leu Leu His AlaGlu 1085 1090 1095 Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met GlyIle Gly 1100 1105 1110 Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala GluPhe Gln Leu 1115 1120 1125 Ser Ala Gly Arg Tyr His Tyr Gln Leu Val TrpCys Gln Lys 1130 1135 1140 16 40 DNA Artificial Sequence assemblingoligo for CpG-Free Sh ble 16 aactcagctg aggaggcaga ccatggccaa gttgaccagt40 17 40 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 17gctgtcccag tgctcacagc cagggatgtg gctggagctg 40 18 40 DNA ArtificialSequence assembling oligo for CpG-Free Sh ble 18 ttgagttctg gactgacaggttggggttct ccagagattt 40 19 40 DNA Artificial Sequence assembling oligofor CpG-Free Sh ble 19 tgtggaggat gactttgcag gtgtggtcag agatgatgtc 40 2040 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 20accctgttca tctcagcagt ccaggaccag gtggtgcctg 40 21 40 DNA ArtificialSequence assembling oligo for CpG-Free Sh ble 21 acaacaccct ggcttgggtgtgggtgagag gactggatga 40 22 40 DNA Artificial Sequence assembling oligofor CpG-Free Sh ble 22 gctgtatgct gagtggagtg aggtggtctc caccaacttc 40 2340 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 23agggatgcca gtggccctgc catgacagag attggagagc 40 24 40 DNA ArtificialSequence assembling oligo for CpG-Free Sh ble 24 agccctgggg gagagagtttgccctgagag acccagcagg 40 25 40 DNA Artificial Sequence assembling oligofor CpG-Free Sh ble 25 caactgtgtg cactttgtgg cagaggagca ggactgagga 40 2621 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 26taagaattca gctagctcga c 21 27 41 DNA Artificial Sequence assemblingoligo for CpG-Free Sh ble 27 gtcgagctag ctgaattctt atcctcagtc ctgctcctctg 41 28 40 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble28 ccacaaagtg cacacagttg cctgctgggt ctctcagggc 40 29 40 DNA ArtificialSequence assembling oligo for CpG-Free Sh ble 29 aaactctctc ccccagggctgctctccaat ctctgtcatg 40 30 40 DNA Artificial Sequence assembling oligofor CpG-Free Sh ble 30 gcagggccac tggcatccct gaagttggtg gagaccacct 40 3140 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 31cactccactc agcatacagc tcatccagtc ctctcaccca 40 32 40 DNA ArtificialSequence assembling oligo for CpG-Free Sh ble 32 cacccaagcc agggtgttgtcaggcaccac ctggtcctgg 40 33 40 DNA Artificial Sequence assembling oligofor CpG-Free Sh ble 33 actgctgaga tgaacagggt gacatcatct ctgaccacac 40 3440 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 34ctgcaaagtc atcctccaca aaatctctgg agaaccccaa 40 35 40 DNA ArtificialSequence assembling oligo for CpG-Free Sh ble 35 cctgtcagtc cagaactcaacagctccagc cacatccctg 40 36 40 DNA Artificial Sequence assembling oligofor CpG-Free Sh ble 36 gctgtgagca ctgggacagc actggtcaac ttggccatgg 40 3720 DNA Artificial Sequence assembling oligo for CpG-Free Sh ble 37tctgcctcct cagctgagtt 20 38 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 38 tgagatcacc ggttcagctg aggaggcaca tcatgaagaaacctgaactg acagcaactt 60 39 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 39 ctgttgagaa gtttctcatt gaaaaatttg attctgtttctgatctcatg cagctgtctg 60 40 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 40 aaggtgaaga aagcagagcc ttttcttttg atgttggaggaagaggttat gttctgaggg 60 41 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 41 tcaattcttg tgctgatggt ttttacaaag acagatatgtttacagacac tttgcctctg 60 42 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 42 ctgctctgcc aattccagaa gttctggaca ttggagaattttctgaatct ctcacctact 60 43 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 43 gcatcagcag aagagcacaa ggagtcactc tccaggatctccctgaaact gagctgccag 60 44 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 44 ctgttctgca acctgttgct gaagcaatgg atgccattgcagcagctgat ctgagccaaa 60 45 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 45 cctctggatt tggtcctttt ggtccccaag gcattggtcagtacaccact tggagggatt 60 46 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 46 tcatttgtgc cattgctgat cctcatgtct atcactggcagactgtgatg gatgacacag 60 47 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 47 tttctgcttc tgttgctcag gcactggatg aactcatgctgtgggcagaa gattgtcctg 60 48 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 48 aagtcagaca cctggtccat gctgattttg gaagcaacaatgttctgaca gacaatggca 60 49 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 49 gaatcactgc agtcattgac tggtctgaag ccatgtttggagattctcaa tatgaggttg 60 50 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 50 ccaacatttt tttttggaga ccttggctgg cttgcatggaacaacaaaca agatattttg 60 51 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 51 aaagaagaca cccagaactg gctggttccc ccagactgagagcctacatg ctcagaattg 60 52 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 52 gcctggacca actgtatcaa tctctggttg atggaaactttgatgatgct gcttgggcac 60 53 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 53 aaggaagatg tgatgccatt gtgaggtctg gtgctggaactgttggaaga actcaaattg 60 54 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 54 caagaaggtc tgctgctgtt tggactgatg gatgtgttgaagttctggct gactctggaa 60 55 60 DNA Artificial Sequence assembling oligofor CpG-Free Hph 55 acaggagacc ctccacaaga cccagagcca aggaatgaatattagctagc ggatcctgag 60 56 30 DNA Artificial Sequence assembling oligofor CpG-Free Hph 56 ctcaggatcc gctagctaat attcattcct 30 57 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 57 tggctctgggtcttgtggag ggtctcctgt ttccagagtc agccagaact tcaacacatc 60 58 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 58 catcagtccaaacagcagca gaccttcttg caatttgagt tcttccaaca gttccagcac 60 59 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 59 cagacctcacaatggcatca catcttcctt gtgcccaagc agcatcatca aagtttccat 60 60 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 60 caaccagagattgatacagt tggtccaggc caattctgag catgtaggct ctcagtctgg 60 61 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 61 gggaaccagccagttctggg tgtcttcttt caaaatatct tgtttgttgt tccatgcaag 60 62 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 62 ccagccaaggtctccaaaaa aaaatgttgg caacctcata ttgagaatct ccaaacatgg 60 63 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 63 cttcagaccagtcaatgact gcagtgattc tgccattgtc tgtcagaaca ttgttgcttc 60 64 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 64 caaaatcagcatggaccagg tgtctgactt caggacaatc ttctgcccac agcatgagtt 60 65 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 65 catccagtgcctgagcaaca gaagcagaaa ctgtgtcatc catcacagtc tgccagtgat 60 66 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 66 agacatgaggatcagcaatg gcacaaatga aatccctcca agtggtgtac tgaccaatgc 60 67 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 67 cttggggaccaaaaggacca aatccagagg tttggctcag atcagctgct gcaatggcat 60 68 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 68 ccattgcttcagcaacaggt tgcagaacag ctggcagctc agtttcaggg agatcctgga 60 69 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 69 gagtgactccttgtgctctt ctgctgatgc agtaggtgag agattcagaa aattctccaa 60 70 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 70 tgtccagaacttctggaatt ggcagagcag cagaggcaaa gtgtctgtaa acatatctgt 60 71 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 71 ctttgtaaaaaccatcagca caagaattga ccctcagaac ataacctctt cctccaacat 60 72 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 72 caaaagaaaaggctctgctt tcttcacctt cagacagctg catgagatca gaaacagaat 60 73 60 DNAArtificial Sequence assembling oligo for CpG-Free Hph 73 caaatttttcaatgagaaac ttctcaacag aagttgctgt cagttcaggt ttcttcatga 60 74 30 DNAArtificial Sequence assembling oligo for CpG-Free Hph 74 tgtgcctcctcagctgaacc ggtgatctca 30 75 40 DNA Artificial Sequence assembling oligofor CpG-Free Bsr 75 aggaggcaca tcatgaagac cttcaacatc tctcagcagg 40 76 40DNA Artificial Sequence assembling oligo for CpG-Free Bsr 76 atctggagctggtggaggtc gccactgaga agatcaccat 40 77 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 77 gctctatgag gacaacaagc accatgtcggggcggccatc 40 78 40 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 78 aggaccaaga ctggggagat catctctgct gtccacattg 40 79 40 DNAArtificial Sequence assembling oligo for CpG-Free Bsr 79 aggcctacattggcagggtc actgtctgtg ctgaagccat 40 80 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 80 tgccattggg tctgctgtga gcaacgggcagaaggacttt 40 81 40 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 81 gacaccattg tggctgtcag gcacccctac tctgatgagg 40 82 40 DNAArtificial Sequence assembling oligo for CpG-Free Bsr 82 tggacagatccatcagggtg gtcagcccct gtggcatgtg 40 83 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 83 cagagagctc atctctgact atgctcctgactgctttgtg 40 84 40 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 84 ctcattgaga tgaatggcaa gctggtcaaa accaccattg 40 85 40 DNAArtificial Sequence assembling oligo for CpG-Free Bsr 85 aggaactcatccccctcaag tacaccagga actaaacctg 40 86 21 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 86 aattcagcta gctcgacatg a 21 87 41DNA Artificial Sequence assembling oligo for CpG-Free Bsr 87 tcatgtcgagctagctgaat tcaggtttag ttcctggtgt a 41 88 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 88 cttgaggggg atgagttcct caatggtggttttgaccagc 40 89 40 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 89 ttgccattca tctcaatgag cacaaagcag tcaggagcat 40 90 40 DNAArtificial Sequence assembling oligo for CpG-Free Bsr 90 agtcagagatgagctctctg cacatgccac aggggctgac 40 91 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 91 caccctgatg gatctgtcca cctcatcagagtaggggtgc 40 92 40 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 92 ctgacagcca caatggtgtc aaagtccttc tgcccgttgc 40 93 40 DNAArtificial Sequence assembling oligo for CpG-Free Bsr 93 tcacagcagacccaatggca atggcttcag cacagacagt 40 94 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 94 gaccctgcca atgtaggcct caatgtggacagcagagatg 40 95 40 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 95 atctccccag tcttggtcct gatggccgcc ccgacatggt 40 96 40 DNAArtificial Sequence assembling oligo for CpG-Free Bsr 96 gcttgttgtcctcatagagc atggtgatct tctcagtggc 40 97 40 DNA Artificial Sequenceassembling oligo for CpG-Free Bsr 97 gacctccacc agctccagat cctgctgagagatgttgaag 40 98 20 DNA Artificial Sequence assembling oligo forCpG-Free Bsr 98 gtcttcatga tgtgcctcct 20 99 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 99 ctcactatag gaggaccatc atgactgagtacaaacccac agtgaggctg gcaaccagag 60 100 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 100 atgatgttcc aagagctgtg agaacactggctgctgcttt tgcagactac cctgcaacaa 60 101 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 101 ggcacacagt tgaccctgac aggcacattgagagggtgac agaactgcaa gaactcttcc 60 102 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 102 tcaccagagt gggactggac attggaaaagtttgggttgc agatgatgga gctgctgttg 60 103 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 103 cagtttggac aacacctgag tctgttgaagctggtgctgt ttttgctgaa attggaccaa 60 104 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 104 gaatggctga gctctctgga agcaggctggcagcacaaca acaaatggaa ggtctgctgg 60 105 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 105 caccacacag gccaaaagag ccagcttggtttctggcaac tgttggagtg agccctgacc 60 106 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 106 accagggaaa gggtctggga tctgctgttgttctgcctgg agttgaagct gctgaaaggg 60 107 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 107 ctggagttcc tgcctttctg gaaacttctgctcccagaaa cctgcctttt tatgaaagac 60 108 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 108 tgggattcac tgtgacagct gatgttgaggttccagaagg cccaagaact tggtgcatga 60 109 60 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 109 caaggaagcc tggagcttaa acctgagctagctcgacatg ataagataca ttgatgagtt 60 110 30 DNA Artificial Sequenceassembling oligo for CpG-Free Pac 110 aactcatcaa tgtatcttat catgtcgagc30 111 60 DNA Artificial Sequence assembling oligo for CpG-Free Pac 111tagctcaggt ttaagctcca ggcttccttg tcatgcacca agttcttggg ccttctggaa 60 11260 DNA Artificial Sequence assembling oligo for CpG-Free Pac 112cctcaacatc agctgtcaca gtgaatccca gtctttcata aaaaggcagg tttctgggag 60 11360 DNA Artificial Sequence assembling oligo for CpG-Free Pac 113cagaagtttc cagaaaggca ggaactccag ccctttcagc agcttcaact ccaggcagaa 60 11460 DNA Artificial Sequence assembling oligo for CpG-Free Pac 114caacagcaga tcccagaccc tttccctggt ggtcagggct cactccaaca gttgccagaa 60 11560 DNA Artificial Sequence assembling oligo for CpG-Free Pac 115accaagctgg ctcttttggc ctgtgtggtg ccagcagacc ttccatttgt tgttgtgctg 60 11660 DNA Artificial Sequence assembling oligo for CpG-Free Pac 116ccagcctgct tccagagagc tcagccattc ttggtccaat ttcagcaaaa acagcaccag 60 11760 DNA Artificial Sequence assembling oligo for CpG-Free Pac 117cttcaacaga ctcaggtgtt gtccaaactg caacagcagc tccatcatct gcaacccaaa 60 11860 DNA Artificial Sequence assembling oligo for CpG-Free Pac 118cttttccaat gtccagtccc actctggtga ggaagagttc ttgcagttct gtcaccctct 60 11960 DNA Artificial Sequence assembling oligo for CpG-Free Pac 119caatgtgcct gtcagggtca actgtgtgcc ttgttgcagg gtagtctgca aaagcagcag 60 12060 DNA Artificial Sequence assembling oligo for CpG-Free Pac 120ccagtgttct cacagctctt ggaacatcat ctctggttgc cagcctcact gtgggtttgt 60 12130 DNA Artificial Sequence assembling oligo for CpG-Free Pac 121actcagtcat gatggtcctc ctatagtgag 30 122 40 DNA Artificial Sequenceassembling oligo for CpG-free LacZ 122 atcactatag gagggccacc atggaccctgttgtgctgca 40 123 20 DNA Artificial Sequence assembling oligo forCpG-free LacZ 123 ggtggccctc ctatagtgat 20 124 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 124 aaggagagac tgggagaaccctggagtgac ccagctcaac 40 125 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 125 ggttctccca gtctctcctt tgcagcacaa cagggtccat 40 12640 DNA Artificial Sequence assembling oligo for CpG-free LacZ 126agactggctg cccaccctcc ctttgcctct tggaggaact 40 127 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 127 ggagggtggg cagccagtctgttgagctgg gtcactccag 40 128 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 128 ctgaggaagc caggacagac aggcccagcc agcagctcag 40 12940 DNA Artificial Sequence assembling oligo for CpG-free LacZ 129gtctgtcctg gcttcctcag agttcctcca agaggcaaag 40 130 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 130 gtctctcaat ggagagtggaggtttgcctg gttccctgcc 40 131 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 131 tccactctcc attgagagac ctgagctgct ggctgggcct 40 13240 DNA Artificial Sequence assembling oligo for CpG-free LacZ 132cctgaagctg tgcctgagtc ttggctggag tgtgacctcc 40 133 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 133 gactcaggca cagcttcaggggcagggaac caggcaaacc 40 134 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 134 cagaggctga cactgttgtg gtgcccagca actggcagat 40 13540 DNA Artificial Sequence assembling oligo for CpG-free LacZ 135cacaacagtg tcagcctctg ggaggtcaca ctccagccaa 40 136 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 136 gcatggctat gatgcccccatctacaccaa tgtcacctac 40 137 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 137 tgggggcatc atagccatgc atctgccagt tgctgggcac 40 13840 DNA Artificial Sequence assembling oligo for CpG-free LacZ 138cccatcactg tgaacccccc ttttgtgccc actgagaacc 40 139 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 139 ggggggttca cagtgatggggtaggtgaca ttggtgtaga 40 140 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 140 ccactggctg ctacagcctg accttcaatg ttgatgagag 40 14140 DNA Artificial Sequence assembling oligo for CpG-free LacZ 141caggctgtag cagccagtgg ggttctcagt gggcacaaaa 40 142 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 142 ctggctgcaa gaaggccagaccaggatcat ctttgatgga 40 143 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 143 tctggccttc ttgcagccag ctctcatcaa cattgaaggt 40 14440 DNA Artificial Sequence assembling oligo for CpG-free LacZ 144gtcaactctg ccttccacct ctggtgcaat ggcaggtggg 40 145 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 145 aggtggaagg cagagttgactccatcaaag atgatcctgg 40 146 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 146 ttggctatgg ccaagacagc aggctgccct ctgagtttga 40 14740 DNA Artificial Sequence assembling oligo for CpG-free LacZ 147gctgtcttgg ccatagccaa cccacctgcc attgcaccag 40 148 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 148 cctctctgcc ttcctcagagctggagagaa caggctggct 40 149 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 149 ctctgaggaa ggcagagagg tcaaactcag agggcagcct 40 15040 DNA Artificial Sequence assembling oligo for CpG-free LacZ 150gtcatggtgc tcaggtggtc tgatggcagc tacctggaag 40 151 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 151 gaccacctga gcaccatgacagccagcctg ttctctccag 40 152 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 152 accaagacat gtggaggatg tctggcatct tcagggatgt 40 15340 DNA Artificial Sequence assembling oligo for CpG-free LacZ 153catcctccac atgtcttggt cttccaggta gctgccatca 40 154 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 154 gagcctgctg cacaagcccaccacccagat ttctgacttc 40 155 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 155 tgggcttgtg cagcaggctc acatccctga agatgccaga 40 15640 DNA Artificial Sequence assembling oligo for CpG-free LacZ 156catgttgcca ccaggttcaa tgatgacttc agcagagctg 40 157 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 157 ttgaacctgg tggcaacatggaagtcagaa atctgggtgg 40 158 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 158 tgctggaggc tgaggtgcag atgtgtggag aactcagaga 40 15940 DNA Artificial Sequence assembling oligo for CpG-free LacZ 159ctgcacctca gcctccagca cagctctgct gaagtcatca 40 160 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 160 ctacctgaga gtcacagtgagcctctggca aggtgagacc 40 161 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 161 tcactgtgac tctcaggtag tctctgagtt ctccacacat 40 16240 DNA Artificial Sequence assembling oligo for CpG-free LacZ 162caggtggcct ctggcacagc cccctttgga ggagagatca 40 163 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 163 gctgtgccag aggccacctgggtctcacct tgccagaggc 40 164 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 164 ttgatgagag aggaggctat gctgacagag tcaccctgag 40 16540 DNA Artificial Sequence assembling oligo for CpG-free LacZ 165atagcctcct ctctcatcaa tgatctctcc tccaaagggg 40 166 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 166 gctcaatgtg gagaaccccaagctgtggtc tgctgagatc 40 167 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 167 tggggttctc cacattgagc ctcagggtga ctctgtcagc 40 16840 DNA Artificial Sequence assembling oligo for CpG-free LacZ 168cccaacctct acagggctgt tgtggagctg cacactgctg 40 169 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 169 acagccctgt agaggttggggatctcagca gaccacagct 40 170 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 170 atggcaccct gattgaagct gaagcctgtg atgttggatt 40 17140 DNA Artificial Sequence assembling oligo for CpG-free LacZ 171agcttcaatc agggtgccat cagcagtgtg cagctccaca 40 172 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 172 cagagaagtc aggattgagaatggcctgct gctgctcaat 40 173 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 173 tctcaatcct gacttctctg aatccaacat cacaggcttc 40 17440 DNA Artificial Sequence assembling oligo for CpG-free LacZ 174ggcaagcctc tgctcatcag gggagtcaac aggcatgagc 40 175 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 175 ctgatgagca gaggcttgccattgagcagc agcaggccat 40 176 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 176 accaccctct gcatggacaa gtgatggatg aacagacaat 40 17740 DNA Artificial Sequence assembling oligo for CpG-free LacZ 177ttgtccatgc agagggtggt gctcatgcct gttgactccc 40 178 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 178 ggtgcaagat atcctgctgatgaagcagaa ctccgcctac 40 179 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 179 tcagcaggat atcttgcacc attgtctgtt catccatcac 40 18020 DNA Artificial Sequence assembling oligo for CpG-free LacZ 180gtaggcggag ttctgcttca 20 181 20 DNA Artificial Sequence assembling oligofor CpG-free LacZ 181 tcattagcag gatatcttgc 20 182 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 182 gcaagatatc ctgctaatgaagcagaacaa cttcaatgct 40 183 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 183 gggtagtgag agcacctgac agcattgaag ttgttctgct 40 18440 DNA Artificial Sequence assembling oligo for CpG-free LacZ 184gtcaggtgct ctcactaccc caaccaccct ctctggtaca 40 185 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 185 gccatacctg tcacacagggtgtaccagag agggtggttg 40 186 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 186 ccctgtgtga caggtatggc ctgtatgttg ttgatgaagc 40 18740 DNA Artificial Sequence assembling oligo for CpG-free LacZ 187tgccatgtgt ctcaatgttg gcttcatcaa caacatacag 40 188 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 188 caacattgag acacatggcatggtgcccat gaacaggctc 40 189 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 189 agccacctgg ggtcatctgt gagcctgttc atgggcacca 40 19040 DNA Artificial Sequence assembling oligo for CpG-free LacZ 190acagatgacc ccaggtggct gcctgccatg tctgagagag 40 191 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 191 tctctgcacc atcctggtcactctctcaga catggcaggc 40 192 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 192 tgaccaggat ggtgcagaga gacaggaacc acccctctgt 40 19340 DNA Artificial Sequence assembling oligo for CpG-free LacZ 193tgcccagaga ccagatgatc acagaggggt ggttcctgtc 40 194 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 194 gatcatctgg tctctgggcaatgagtctgg acatggagcc 40 195 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 195 ctgtagagag catcatggtt ggctccatgt ccagactcat 40 19640 DNA Artificial Sequence assembling oligo for CpG-free LacZ 196aaccatgatg ctctctacag gtggatcaag tctgttgacc 40 197 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 197 atactgcaca ggtctgctggggtcaacaga cttgatccac 40 198 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 198 ccagcagacc tgtgcagtat gaaggaggtg gagcagacac 40 19940 DNA Artificial Sequence assembling oligo for CpG-free LacZ 199agatgatgtc tgtggctgtg gtgtctgctc cacctccttc 40 200 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 200 cacagccaca gacatcatctgccccatgta tgccagggtt 40 201 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 201 gggaagggct ggtcctcatc aaccctggca tacatggggc 40 20240 DNA Artificial Sequence assembling oligo for CpG-free LacZ 202gatgaggacc agcccttccc tgctgtgccc aagtggagca 40 203 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 203 cagagagagc cacttcttgatgctccactt gggcacagca 40 204 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 204 tcaagaagtg gctctctctg cctggagaga ccagacctct 40 20540 DNA Artificial Sequence assembling oligo for CpG-free LacZ 205gtgcatattc acacaggatc agaggtctgg tctctccagg 40 206 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 206 gatcctgtgt gaatatgcacatgcaatggg caactctctg 40 207 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 207 cagtacttgg caaagcctcc cagagagttg cccattgcat 40 20840 DNA Artificial Sequence assembling oligo for CpG-free LacZ 208ggaggctttg ccaagtactg gcaagccttc agacagtacc 40 209 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 209 aaatcctcct tgcagcctggggtactgtct gaaggcttgc 40 210 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 210 ccaggctgca aggaggattt gtgtgggact gggtggacca 40 21140 DNA Artificial Sequence assembling oligo for CpG-free LacZ 211catcatactt gatgagagat tggtccaccc agtcccacac 40 212 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 212 atctctcatc aagtatgatgagaatggcaa cccctggtct 40 213 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 213 ccaaagtctc ctccataggc agaccagggg ttgccattct 40 21440 DNA Artificial Sequence assembling oligo for CpG-free LacZ 214gcctatggag gagactttgg tgacaccccc aatgacaggc 40 215 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 215 caggccattc atgcagaactgcctgtcatt gggggtgtca 40 216 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 216 agttctgcat gaatggcctg gtctttgcag acaggacccc 40 21740 DNA Artificial Sequence assembling oligo for CpG-free LacZ 217cctctgtgag ggcagggtga ggggtcctgt ctgcaaagac 40 218 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 218 tcaccctgcc ctcacagaggccaagcacca gcaacagttc 40 219 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 219 ccagacagcc tgaactggaa gaactgttgc tggtgcttgg 40 22040 DNA Artificial Sequence assembling oligo for CpG-free LacZ 220ttccagttca ggctgtctgg acagaccatt gaggtgacat 40 221 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 221 gtgcctgaag aggtactcagatgtcacctc aatggtctgt 40 222 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 222 ctgagtacct cttcaggcac tctgacaatg agctcctgca 40 22320 DNA Artificial Sequence assembling oligo for CpG-free LacZ 223tgcaggagct cattgtcaga 20 224 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 224 gtaatttaac aatgagctcc tgcactggat ggtggccctg 40 22520 DNA Artificial Sequence assembling oligo for CpG-free LacZ 225ggagctcatt gttaaattac 20 226 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 226 gatggcaagc ctctggcttc tggtgaggtg cctctggatg 40 22740 DNA Artificial Sequence assembling oligo for CpG-free LacZ 227gaagccagag gcttgccatc cagggccacc atccagtgca 40 228 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 228 tggcccctca aggaaagcagctgattgaac tgcctgagct 40 229 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 229 ctgctttcct tgaggggcca catccagagg cacctcacca 40 23040 DNA Artificial Sequence assembling oligo for CpG-free LacZ 230gcctcagcca gagtctgctg gacaactgtg gctaacagtg 40 231 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 231 cagcagactc tggctgaggcagctcaggca gttcaatcag 40 232 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 232 agggtggttc agcccaatgc aacagcttgg tctgaggcag 40 23340 DNA Artificial Sequence assembling oligo for CpG-free LacZ 233gcattgggct gaaccaccct cactgttagc cacagttgtc 40 234 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 234 gccacatctc tgcatggcagcagtggaggc tggctgagaa 40 235 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 235 ctgccatgca gagatgtggc ctgcctcaga ccaagctgtt 40 23640 DNA Artificial Sequence assembling oligo for CpG-free LacZ 236cctctctgtg accctgcctg ctgcctctca tgccatccct 40 237 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 237 caggcagggt cacagagaggttctcagcca gcctccactg 40 238 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 238 cacctgacaa catctgaaat ggacttctgc attgagctgg 40 23940 DNA Artificial Sequence assembling oligo for CpG-free LacZ 239atttcagatg ttgtcaggtg agggatggca tgagaggcag 40 240 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 240 gcaacaagag atggcagttcaacaggcagt ctggcttcct 40 241 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 241 gaactgccat ctcttgttgc ccagctcaat gcagaagtcc 40 24240 DNA Artificial Sequence assembling oligo for CpG-free LacZ 242gtctcagatg tggattggag acaagaagca gctcctcacc 40 243 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 243 ctccaatcca catctgagacaggaagccag actgcctgtt 40 244 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 244 cctctcaggg accaattcac cagggctcct ctggacaatg 40 24540 DNA Artificial Sequence assembling oligo for CpG-free LacZ 245gtgaattggt ccctgagagg ggtgaggagc tgcttcttgt 40 246 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 246 acattggagt gtctgaggccaccaggattg acccaaatgc 40 247 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 247 ggcctcagac actccaatgt cattgtccag aggagccctg 40 24840 DNA Artificial Sequence assembling oligo for CpG-free LacZ 248ttgggtggag aggtggaagg ctgctggaca ctaccaggct 40 249 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 249 ccttccacct ctccacccaagcatttgggt caatcctggt 40 250 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 250 gaggctgccc tgctccagtg cacagcagac accctggctg 40 25140 DNA Artificial Sequence assembling oligo for CpG-free LacZ 251cactggagca gggcagcctc agcctggtag tgtccagcag 40 252 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 252 atgctgttct gatcaccacagcccatgctt ggcagcacca 40 253 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 253 tgtggtgatc agaacagcat cagccagggt gtctgctgtg 40 25440 DNA Artificial Sequence assembling oligo for CpG-free LacZ 254aggcaagacc ctgttcatca gcagaaagac ctacaggatt 40 255 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 255 tgatgaacag ggtcttgccttggtgctgcc aagcatgggc 40 256 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 256 gatggctctg gacagatggc aatcacagtg gatgtggagg 40 25740 DNA Artificial Sequence assembling oligo for CpG-free LacZ 257gccatctgtc cagagccatc aatcctgtag gtctttctgc 40 258 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 258 ttgcctctga cacacctcaccctgcaagga ttggcctgaa 40 259 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 259 gtgaggtgtg tcagaggcaa cctccacatc cactgtgatt 40 26040 DNA Artificial Sequence assembling oligo for CpG-free LacZ 260ctgtcaactg gcacaggtgg ctgagagggt gaactggctg 40 261 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 261 ccacctgtgc cagttgacagttcaggccaa tccttgcagg 40 262 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 262 ggcttaggcc ctcaggagaa ctaccctgac aggctgacag 40 26340 DNA Artificial Sequence assembling oligo for CpG-free LacZ 263ttctcctgag ggcctaagcc cagccagttc accctctcag 40 264 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 264 ctgcctgctt tgacaggtgggacctgcctc tgtctgacat 40 265 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 265 ccacctgtca aagcaggcag ctgtcagcct gtcagggtag 40 26640 DNA Artificial Sequence assembling oligo for CpG-free LacZ 266gtacacccct tatgtgttcc cttctgagaa tggcctgagg 40 267 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 267 ggaacacata aggggtgtacatgtcagaca gaggcaggtc 40 268 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 268 tgtggcacca gggagctgaa ctatggtcct caccagtgga 40 26940 DNA Artificial Sequence assembling oligo for CpG-free LacZ 269ttcagctccc tggtgccaca cctcaggcca ttctcagaag 40 270 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 270 ggggagactt ccagttcaacatctccaggt actctcagca 40 271 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 271 gttgaactgg aagtctcccc tccactggtg aggaccatag 40 27240 DNA Artificial Sequence assembling oligo for CpG-free LacZ 272acagctcatg gaaacctctc acaggcacct gctccatgca 40 273 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 273 gagaggtttc catgagctgttgctgagagt acctggagat 40 274 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 274 gaggagggaa cctggctgaa cattgatggc ttccacatgg 40 27540 DNA Artificial Sequence assembling oligo for CpG-free LacZ 275ttcagccagg ttccctcctc tgcatggagc aggtgcctgt 40 276 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 276 gcattggagg agatgactcttggtctcctt ctgtgtctgc 40 277 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 277 agagtcatct cctccaatgc ccatgtggaa gccatcaatg 40 27840 DNA Artificial Sequence assembling oligo for CpG-free LacZ 278tgagttccag ttatctgctg gcaggtacca ctatcagctg 40 279 40 DNA ArtificialSequence assembling oligo for CpG-free LacZ 279 cagcagataa ctggaactcagcagacacag aaggagacca 40 280 40 DNA Artificial Sequence assembling oligofor CpG-free LacZ 280 gtgtggtgcc agaagtaaac ctgagctagc agtccatgat 40 28140 DNA Artificial Sequence assembling oligo for CpG-free LacZ 281gtttacttct ggcaccacac cagctgatag tggtacctgc 40 282 20 DNA ArtificialSequence assembling oligo for CpG-free LacZ 282 atcatggact gctagctcag 20283 40 DNA Artificial Sequence assembling oligo for R6K gamma M2Areplication origin 283 gcaggactga ggcttaatta aaccttaaaa cctttaaaag 40284 40 DNA Artificial Sequence assembling oligo for R6K gamma M2Areplication origin 284 ccttatatat tctttttttt cttataaaac ttaaaacctt 40285 40 DNA Artificial Sequence assembling oligo for R6K gamma M2Areplication origin 285 agaggctatt taagttgctg atttatatta attttattgt 40286 50 DNA Artificial Sequence assembling oligo for R6K gamma M2Areplication origin 286 tcaaacatga gagcttagta catgaaacat gagagcttagtacattagcc 50 287 60 DNA Artificial Sequence assembling oligo for R6Kgamma M2A replication origin 287 atgagagctt agtacattag ccatgagggtttagttcatt aaacatgaga gcttagtaca 60 288 60 DNA Artificial Sequenceassembling oligo for R6K gamma M2A replication origin 288 ttaaacatgagagcttagta catgaaacat gagagcttag tacatactat caacaggttg 60 289 30 DNAArtificial Sequence assembling oligo for R6K gamma M2A replicationorigin 289 aactgctgat cttaattaac ctggagactt 30 290 60 DNA ArtificialSequence assembling oligo for R6K gamma M2A replication origin 290aagtctccag gttaattaag atcagcagtt caacctgttg atagtatgta ctaagctctc 60 29160 DNA Artificial Sequence assembling oligo for R6K gamma M2Areplication origin 291 atgtttcatg tactaagctc tcatgtttaa tgtactaagctctcatgttt aatgaactaa 60 292 60 DNA Artificial Sequence assembling oligofor R6K gamma M2A replication origin 292 accctcatgg ctaatgtactaagctctcat ggctaatgta ctaagctctc atgtttcatg 60 293 40 DNA ArtificialSequence assembling oligo for R6K gamma M2A replication origin 293tactaagctc tcatgtttga acaataaaat taatataaat 40 294 40 DNA ArtificialSequence assembling oligo for R6K gamma M2A replication origin 294cagcaactta aatagcctct aaggttttaa gttttataag 40 295 40 DNA ArtificialSequence assembling oligo for R6K gamma M2A replication origin 295aaaaaaaaga atatataagg cttttaaagg ttttaaggtt 40 296 20 DNA ArtificialSequence assembling oligo for R6K gamma M2A replication origin 296taattaagcc tcagtcctgc 20 297 66 DNA Artificial Sequence EM7 promotor 297caattaatca tcggcatagt atatcggcat agtataatac gactcactat aggagggcca 60ccatgg 66 298 66 DNA Artificial Sequence degenerated oligo forassembling the GpG-Free EM2K promotor 298 caattaawca tdggcatagtatatcwgcat agtataatac hactcactat aggagggcca 60 ccatgg 66 299 20 DNAArtificial Sequence sense oligo for the assembly of SEQ ID NO 38-74oligos 299 ttcagctgag gaggcacatc 20 300 20 DNA Artificial Sequencereverse oligo for the assembly of SEQ ID NO 38-74 oligos 300 ctcaggatccgctagctaat 20 301 18 DNA Artificial Sequence sense primer pur 24 301aggaccatca tgactgag 18 302 18 DNA Artificial Sequence reverse primer pur25 302 atcatgtcga gctagctc 18 303 31 DNA Artificial Sequence RK15 primer303 gcaggactga ggcttaatta aaccttaaaa c 31 304 31 DNA Artificial SequenceRK16 primer 304 aagtctccag gttaattaag atcagcagtt c 31 305 52 DNAArtificial Sequence rps0-1 linker oligo 305 ctagctgagt ttcagaaaagggggcctgag tggccccttt tttcaactta at 52 306 46 DNA Artificial Sequencerps0-2 linker oligo 306 taagttgaaa aaaggggcca ctcaggcccc cttttctgaaactcag 46 307 39 DNA Artificial Sequence PACI-UP primer 307 atcgttaattaaaacagtag ttgacaatta aacattggc 39 308 28 DNA Artificial SequencePACI-DOWN primer 308 atcgttaatt aagttgaaaa aaggggcc 28 309 17 DNAArtificial Sequence rbs-1 linker oligo 309 aattctgagg agaagct 17 310 17DNA Artificial Sequence rbs-2 linker oligo 310 catgagcttc tcctcag 17 31136 DNA Artificial Sequence OL dcm AF primer 311 ttttgcggcc gcttgctgcgccagcaacta ataacg 36 312 41 DNA Artificial Sequence OL dcm AR primer 312ccttggatcc tggtaaacac gcactgtccg ccaatcgatt c 41 313 30 DNA ArtificialSequence OL dcm BF primer 313 ttttggatcc tcagcaagag gcacaacatg 30 314 35DNA Artificial Sequence OL dcm BR primer 314 ttttctcgag aaacggcagctctgatactt gcttc 35 315 32 DNA Artificial Sequence OL dcm CF primer 315ttttgcggcc gcgttgcggt attacccttg tc 32 316 795 DNA Artificial SequenceNeo DeltaCpG 316 atg att gaa caa gat ggc cta cat gca ggt tct cca gct gcctgg gtt 48 Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala TrpVal 1 5 10 15 gag aga ctg ttt ggc tat gac tgg gca cag cag acc att ggttgc tct 96 Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly CysSer 20 25 30 gat gca gca gtt ttc aga ctt tca gcc caa ggc agg cca gtc cttttt 144 Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe35 40 45 gta aag aca gac ctc agt ggg gct ctc aat gag ctc cag gat gag gct192 Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 5055 60 gcc aga ctc tcc tgg ttg gca aca act ggg gtc ccc tgt gca gct gtc240 Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 6570 75 80 ctt gat gtg gtc aca gaa gct gga agg gac tgg ctc cta cta ggt gag288 Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 8590 95 gtg cct ggg cag gac ctc ctt tcc tct cac cta gct cca gct gag aaa336 Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys 100105 110 gtg tca atc atg gct gat gcc atg aga aga ctc cac acc ctt gac cca384 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro 115120 125 gcc acc tgc ccc ttt gac cac cag gcc aag cac agg ata gag agg gcc432 Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala 130135 140 aga acc agg atg gag gct ggc ctg gtg gac caa gat gac ttg gat gaa480 Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu 145150 155 160 gaa cac cag ggc ctg gcc cct gct gaa cta ttt gcc agg ctc aaggca 528 Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala165 170 175 tcc atg cca gat ggt gag gac cta gtg gtg act cat ggg gat gcctgc 576 Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys180 185 190 ctt ccc aac atc atg gtt gaa aat gga agg ttc tct ggc ttc atagac 624 Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp195 200 205 tgt ggc agg ctg gga gtg gct gac agg tac cag gac att gcc ctagca 672 Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala210 215 220 acc agg gac ata gca gaa gag cta ggg gga gag tgg gca gac aggttc 720 Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe225 230 235 240 cta gtg ctc tat ggc att gca gcc cct gac tcc cag aga attgcc ttc 768 Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile AlaPhe 245 250 255 tac aga ctt ctt gat gag ttc ttc taa 795 Tyr Arg Leu LeuAsp Glu Phe Phe 260 317 264 PRT Artificial Sequence Neo DeltaCpG 317 MetIle Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val 1 5 10 15Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser 20 25 30Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe 35 40 45Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 50 55 60Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 65 70 7580 Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 85 9095 Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys 100105 110 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro115 120 125 Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu ArgAla 130 135 140 Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp LeuAsp Glu 145 150 155 160 Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe AlaArg Leu Lys Ala 165 170 175 Ser Met Pro Asp Gly Glu Asp Leu Val Val ThrHis Gly Asp Ala Cys 180 185 190 Leu Pro Asn Ile Met Val Glu Asn Gly ArgPhe Ser Gly Phe Ile Asp 195 200 205 Cys Gly Arg Leu Gly Val Ala Asp ArgTyr Gln Asp Ile Ala Leu Ala 210 215 220 Thr Arg Asp Ile Ala Glu Glu LeuGly Gly Glu Trp Ala Asp Arg Phe 225 230 235 240 Leu Val Leu Tyr Gly IleAla Ala Pro Asp Ser Gln Arg Ile Ala Phe 245 250 255 Tyr Arg Leu Leu AspGlu Phe Phe 260 318 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 318 cattaccggt aggcacatca tgattgaaca agatggccta 40 319 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 319catgcaggtt ctccagctgc ctgggttgag agactgtttg 40 320 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 320 gctatgactgggcacagcag accattggtt gctctgatgc 40 321 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 321 agcagttttc agactttcag cccaaggcaggccagtcctt 40 322 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 322 tttgtaaaga cagacctcag tggggctctc aatgagctcc 40 323 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 323aggatgaggc tgccagactc tcctggttgg caacaactgg 40 324 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 324 ggtcccctgtgcagctgtcc ttgatgtggt cacagaagct 40 325 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 325 ggaagggact ggctcctact aggtgaggtgcctgggcagg 40 326 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 326 acctcctttc ctctcaccta gctccagctg agaaagtgtc 40 327 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 327aatcatggct gatgccatga gaagactcca cacccttgac 40 328 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 328 ccagccacctgcccctttga ccaccaggcc aagcacagga 40 329 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 329 tagagagggc cagaaccagg atggaggctggcctggtgga 40 330 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 330 ccaagatgac ttggatgaag aacaccaggg cctggcccct 40 331 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 331gctgaactat ttgccaggct caaggcatcc atgccagatg 40 332 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 332 gtgaggacctagtggtgact catggggatg cctgccttcc 40 333 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 333 caacatcatg gttgaaaatg gaaggttctctggcttcata 40 334 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 334 gactgtggca ggctgggagt ggctgacagg taccaggaca 40 335 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 335ttgccctagc aaccagggac atagcagaag agctaggggg 40 336 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 336 agagtgggcagacaggttcc tagtgctcta tggcattgca 40 337 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 337 gcccctgact cccagagaat tgccttctacagacttcttg 40 338 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 338 atgagttctt ctaaagctag ctgatcctga tagctgttcg 40 339 20DNA Artificial Sequence oligo for construction of Neo DeltaCpG 339cgaacagcta tcaggatcag 20 340 40 DNA Artificial Sequence oligo forconstruction of Neo DeltaCpG 340 ctagctttag aagaactcat caagaagtctgtagaaggca 40 341 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 341 attctctggg agtcaggggc tgcaatgcca tagagcacta 40 342 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 342ggaacctgtc tgcccactct ccccctagct cttctgctat 40 343 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 343 gtccctggttgctagggcaa tgtcctggta cctgtcagcc 40 344 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 344 actcccagcc tgccacagtc tatgaagccagagaaccttc 40 345 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 345 cattttcaac catgatgttg ggaaggcagg catccccatg 40 346 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 346agtcaccact aggtcctcac catctggcat ggatgccttg 40 347 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 347 agcctggcaaatagttcagc aggggccagg ccctggtgtt 40 348 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 348 cttcatccaa gtcatcttgg tccaccaggccagcctccat 40 349 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 349 cctggttctg gccctctcta tcctgtgctt ggcctggtgg 40 350 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 350tcaaaggggc aggtggctgg gtcaagggtg tggagtcttc 40 351 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 351 tcatggcatcagccatgatt gacactttct cagctggagc 40 352 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 352 taggtgagag gaaaggaggt cctgcccaggcacctcacct 40 353 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 353 agtaggagcc agtcccttcc agcttctgtg accacatcaa 40 354 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 354ggacagctgc acaggggacc ccagttgttg ccaaccagga 40 355 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 355 gagtctggcagcctcatcct ggagctcatt gagagcccca 40 356 40 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 356 ctgaggtctg tctttacaaa aaggactggcctgccttggg 40 357 40 DNA Artificial Sequence oligo for construction ofNeo DeltaCpG 357 ctgaaagtct gaaaactgct gcatcagagc aaccaatggt 40 358 40DNA Artificial Sequence oligo for construction of Neo DeltaCpG 358ctgctgtgcc cagtcatagc caaacagtct ctcaacccag 40 359 40 DNA ArtificialSequence oligo for construction of Neo DeltaCpG 359 gcagctggagaacctgcatg taggccatct tgttcaatca 40 360 20 DNA Artificial Sequence oligofor construction of Neo DeltaCpG 360 tgatgtgcct accggtaatg 20

1-27. (Cancelled)
 28. A method for producing a plasmid which is a vectorfor at least one gene, and which is completely devoid of CpG, wherein aplasmid is constructed by assembling, by enzyme ligation, DNA fragments,all devoid of CpG, corresponding to an origin of replication of theplasmid and to the elements constituting a transcriptional unit for saidat least one gene, and this plasmid is transferred into an Escherichiacoli strain expressing the pi protein for replication of the plasmid.29. The method as claimed in claim 28, wherein the origin of replicationof the plasmid is the R6K gamma origin of replication modified byelimination of the CpGs.
 30. An origin of replication for a plasmid,wherein its sequence corresponds to that of the R6K gamma origin ofreplication in which each G of the CpGs of the repeat region of the corehas been replaced with an A, a C or a T, or each C of the CpGs has beenreplaced with a G, an A or a T.
 31. The origin of replication as claimedin claim 30, wherein its sequence comprises the sequence SEQ ID NO: 12or the sequence SEQ ID NO:
 13. 32. The origin of replication as claimedin claim 30, wherein the pi protein-binding sequence is repeated 5 or 6times.
 33. The method as claimed in claim 28, wherein thetranscriptional unit comprises a bacterial promoter devoid of CpG.
 34. Apromoter, whose sequence comprises the sequence SEQ ID NO:
 11. 35. Themethod as claimed in claim 28, wherein the transcriptional unitcomprises a bacterial transcription terminator devoid of CpG.
 36. Themethod as claimed in claim 28, wherein the transcriptional unitcomprises a resistance gene devoid of CpG.
 37. The method as claimed inclaim 28, wherein the transcriptional unit comprises a reporter genedevoid of CpG.
 38. A method for producing a gene which is devoid of CpGwhile at the same time able to be expressed in E. coli, wherein apolynucleotide chain is synthesized by following the amino acid chain ofa protein which can be expressed by E. coli, and assigning to each aminoacid a nucleotide codon selected from those which, according to thegenetic code, and taking into account the degeneracy of this code,correspond to this amino acid, while at the same time eliminating fromthis selection: the codons ACG (Thr), CCG (Pro), GCG (Ala), TCG (Ser),CGA (Arg), CGC (Arg), CGG (Arg), CGT (Arg) containing a CpG, and thecodons which finish with a C when the codon which directly follows itbegins with a G.
 39. The method as claimed in claim 38, wherein thecodons ATA (lie), CTA (Leu), GTA (Val) and TTA (Leu) are also eliminatedfrom said selection.
 40. A gene of at least 250 bp, which can beobtained using the method claimed in claim
 38. 41. A gene, the sequenceof which comprises the sequence SEQ ID NO: 1 from position 3 to position374, the sequence SEQ ID NO: 3 from position 3 to position 1025, thesequence SEQ ID NO: 5 from position 3 to position 422, the sequence SEQID NO: 7 from position 3 to position
 599. 42. A gene, the sequence ofwhich comprises the sequence SEQ ID NO: 9 from position 3 to position3056.
 43. A gene, the sequence of which comprises the sequence SEQ IDNO:
 316. 44. A plasmid comprising an origin of replication as claimed inclaim
 30. 45. The plasmid comprising an origin of replication for aplasmid, wherein its sequence corresponds to that of the R6K gammaorigin of replication in which each G of the CpGs of the repeat regionof the core has been replaced with an A, a C or a T, or each C of theCpGs has been replaced with a G, an A or a T, and a gene of at least 250bp, which can be obtained using the method of claim
 38. 46. The plasmidas claimed in claim 40, further comprising a promoter whose sequencecomprises the sequence SEQ ID NO:
 11. 47. The plasmid as claimed inclaim 44, further comprising a CpG-free transcription terminator. 48.The plasmid as claimed in claim 44, being completely devoid of CpG. 49.A plasmid of SEQ ID NO:
 14. 50. A method for producing a plasmidcompletely devoid of CpG and free of methylation on cytosine in thenucleic acid context CC(A/T)GG, wherein a plasmid as claimed in claim 44is produced by replication in an Escherichia coli strain expressing thepi protein, which is deficient for the dcm methylation system.
 51. AnEscherichia coli cell transformed with the plasmids as claimed in claim44.
 52. The transformed Escherichia coli cell as claimed in claim 51,wherein it expresses a gene encoding a pi protein.
 53. The transformedEscherichia coli cell as claimed in claim 52, which further comprisingan inactivated dcm gene.
 54. A kit for producing plasmids, comprising atleast one cell as claimed in claim 51.